Meta-regression analysis of commensal and pathogenic Escherichia coli survival in soil and water.

PubMed

Franz, Eelco; Schijven, Jack; de Roda Husman, Ana Maria; Blaak, Hetty

2014-06-17

The extent to which pathogenic and commensal E. coli (respectively PEC and CEC) can survive, and which factors predominantly determine the rate of decline, are crucial issues from a public health point of view. The goal of this study was to provide a quantitative summary of the variability in E. coli survival in soil and water over a broad range of individual studies and to identify the most important sources of variability. To that end, a meta-regression analysis on available literature data was conducted. The considerable variation in reported decline rates indicated that the persistence of E. coli is not easily predictable. The meta-analysis demonstrated that for soil and water, the type of experiment (laboratory or field), the matrix subtype (type of water and soil), and temperature were the main factors included in the regression analysis. A higher average decline rate in soil of PEC compared with CEC was observed. The regression models explained at best 57% of the variation in decline rate in soil and 41% of the variation in decline rate in water. This indicates that additional factors, not included in the current meta-regression analysis, are of importance but rarely reported. More complete reporting of experimental conditions may allow future inference on the global effects of these variables on the decline rate of E. coli.

9 CFR 381.94 - Contamination with Microorganisms; process control verification criteria and testing; pathogen...

Code of Federal Regulations, 2010 CFR

2010-01-01

...) Analysis of samples. Laboratories may use any quantitative method for analysis of E. coli that is approved... FSIS in its nationwide microbiological baseline data collection programs and surveys. (Copies of... Surveys used in determining the prevalence of Salmonella on raw products are available in the FSIS Docket...

9 CFR 381.94 - Contamination with Microorganisms; process control verification criteria and testing; pathogen...

Code of Federal Regulations, 2011 CFR

2011-01-01

...) Analysis of samples. Laboratories may use any quantitative method for analysis of E. coli that is approved... FSIS in its nationwide microbiological baseline data collection programs and surveys. (Copies of... Surveys used in determining the prevalence of Salmonella on raw products are available in the FSIS Docket...

9 CFR 381.94 - Contamination with Microorganisms; process control verification criteria and testing; pathogen...

Code of Federal Regulations, 2014 CFR

2014-01-01

...) Analysis of samples. Laboratories may use any quantitative method for analysis of E. coli that is approved... FSIS in its nationwide microbiological baseline data collection programs and surveys. (Copies of... Surveys used in determining the prevalence of Salmonella on raw products are available in the FSIS Docket...

9 CFR 381.94 - Contamination with Microorganisms; process control verification criteria and testing; pathogen...

Code of Federal Regulations, 2012 CFR

2012-01-01

...) Analysis of samples. Laboratories may use any quantitative method for analysis of E. coli that is approved... FSIS in its nationwide microbiological baseline data collection programs and surveys. (Copies of... Surveys used in determining the prevalence of Salmonella on raw products are available in the FSIS Docket...

9 CFR 381.94 - Contamination with Microorganisms; process control verification criteria and testing; pathogen...

Code of Federal Regulations, 2013 CFR

2013-01-01

...) Analysis of samples. Laboratories may use any quantitative method for analysis of E. coli that is approved... FSIS in its nationwide microbiological baseline data collection programs and surveys. (Copies of... Surveys used in determining the prevalence of Salmonella on raw products are available in the FSIS Docket...

Epidemic potential of Escherichia coli ST131 and Klebsiella pneumoniae ST258: a systematic review and meta-analysis

PubMed Central

Dautzenberg, M J D; Haverkate, M R; Bonten, M J M; Bootsma, M C J

2016-01-01

Objectives Observational studies have suggested that Escherichia coli sequence type (ST) 131 and Klebsiella pneumoniae ST258 have hyperendemic properties. This would be obvious from continuously high incidence and/or prevalence of carriage or infection with these bacteria in specific patient populations. Hyperendemicity could result from increased transmissibility, longer duration of infectiousness, and/or higher pathogenic potential as compared with other lineages of the same species. The aim of our research is to quantitatively estimate these critical parameters for E. coli ST131 and K. pneumoniae ST258, in order to investigate whether E. coli ST131 and K. pneumoniae ST258 are truly hyperendemic clones. Primary outcome measures A systematic literature search was performed to assess the evidence of transmissibility, duration of infectiousness, and pathogenicity for E. coli ST131 and K. pneumoniae ST258. Meta-regression was performed to quantify these characteristics. Results The systematic literature search yielded 639 articles, of which 19 data sources provided information on transmissibility (E. coli ST131 n=9; K. pneumoniae ST258 n=10)), 2 on duration of infectiousness (E. coli ST131 n=2), and 324 on pathogenicity (E. coli ST131 n=285; K. pneumoniae ST258 n=39). Available data on duration of carriage and on transmissibility were insufficient for quantitative assessment. In multivariable meta-regression E. coli isolates causing infection were associated with ST131, compared to isolates only causing colonisation, suggesting that E. coli ST131 can be considered more pathogenic than non-ST131 isolates. Date of isolation, location and resistance mechanism also influenced the prevalence of ST131. E. coli ST131 was 3.2 (95% CI 2.0 to 5.0) times more pathogenic than non-ST131. For K. pneumoniae ST258 there were not enough data for meta-regression assessing the influence of colonisation versus infection on ST258 prevalence. Conclusions With the currently available data, it cannot be confirmed nor rejected, that E. coli ST131 or K. pneumoniae ST258 are hyperendemic clones. PMID:26988349

Quantitative contamination assessment of Escherichia coli in baby spinach primary production in Spain: Effects of weather conditions and agricultural practices.

PubMed

Allende, Ana; Castro-Ibáñez, Irene; Lindqvist, Roland; Gil, María Isabel; Uyttendaele, Mieke; Jacxsens, Liesbeth

2017-09-18

A quantitative microbial contamination model of Escherichia coli during primary production of baby spinach was developed. The model included only systematic contamination routes (e.g. soil and irrigation water) and it was used to evaluate the potential impact of weather conditions, agricultural practices as well as bacterial fitness in soil on the E. coli levels present in the crop at harvest. The model can be used to estimate E. coli contamination of baby spinach via irrigation water, via soil splashing due to irrigation water or rain events, and also including the inactivation of E. coli on plants due to solar radiation during a variable time of culturing before harvest. Seasonality, solar radiation and rainfall were predicted to have an important impact on the E. coli contamination. Winter conditions increased E. coli prevalence and levels when compared to spring conditions. As regards agricultural practices, both water quality and irrigation system slightly influenced E. coli levels on baby spinach. The good microbiological quality of the irrigation water (average E. coli counts in positive water samples below 1 log/100mL) could have influenced the differences observed among the tested agricultural practices (water treatment and irrigation system). This quantitative microbial contamination model represents a preliminary framework that assesses the potential impact of different factors and intervention strategies affecting E. coli concentrations at field level. Taking into account that E. coli strains may serve as a surrogate organism for enteric bacterial pathogens, obtained results on E. coli levels on baby spinach may be indicative of the potential behaviour of these pathogens under defined conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

A New Algorithm Using Cross-Assignment for Label-Free Quantitation with LC/LTQ-FT MS

PubMed Central

Andreev, Victor P.; Li, Lingyun; Cao, Lei; Gu, Ye; Rejtar, Tomas; Wu, Shiaw-Lin; Karger, Barry L.

2008-01-01

A new algorithm is described for label-free quantitation of relative protein abundances across multiple complex proteomic samples. Q-MEND is based on the denoising and peak picking algorithm, MEND, previously developed in our laboratory. Q-MEND takes advantage of the high resolution and mass accuracy of the hybrid LTQFT MS mass spectrometer (or other high resolution mass spectrometers, such as a Q-TOF MS). The strategy, termed “cross-assignment”, is introduced to increase substantially the number of quantitated proteins. In this approach, all MS/MS identifications for the set of analyzed samples are combined into a master ID list, and then each LC/MS run is searched for the features that can be assigned to a specific identification from that master list. The reliability of quantitation is enhanced by quantitating separately all peptide charge states, along with a scoring procedure to filter out less reliable peptide abundance measurements. The effectiveness of Q-MEND is illustrated in the relative quantitative analysis of E.coli samples spiked with known amounts of non-E.coli protein digests. A mean quantitation accuracy of 7% and mean precision of 15% is demonstrated. Q-MEND can perform relative quantitation of a set of LC/MS datasets without manual intervention and can generate files compatible with the Guidelines for Proteomic Data Publication. PMID:17441747

A new algorithm using cross-assignment for label-free quantitation with LC-LTQ-FT MS.

PubMed

Andreev, Victor P; Li, Lingyun; Cao, Lei; Gu, Ye; Rejtar, Tomas; Wu, Shiaw-Lin; Karger, Barry L

2007-06-01

A new algorithm is described for label-free quantitation of relative protein abundances across multiple complex proteomic samples. Q-MEND is based on the denoising and peak picking algorithm, MEND, previously developed in our laboratory. Q-MEND takes advantage of the high resolution and mass accuracy of the hybrid LTQ-FT MS mass spectrometer (or other high-resolution mass spectrometers, such as a Q-TOF MS). The strategy, termed "cross-assignment", is introduced to increase substantially the number of quantitated proteins. In this approach, all MS/MS identifications for the set of analyzed samples are combined into a master ID list, and then each LC-MS run is searched for the features that can be assigned to a specific identification from that master list. The reliability of quantitation is enhanced by quantitating separately all peptide charge states, along with a scoring procedure to filter out less reliable peptide abundance measurements. The effectiveness of Q-MEND is illustrated in the relative quantitative analysis of Escherichia coli samples spiked with known amounts of non-E. coli protein digests. A mean quantitation accuracy of 7% and mean precision of 15% is demonstrated. Q-MEND can perform relative quantitation of a set of LC-MS data sets without manual intervention and can generate files compatible with the Guidelines for Proteomic Data Publication.

Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level.

PubMed

Zhu, Zhi; Zhang, Wenhua; Leng, Xuefei; Zhang, Mingxia; Guan, Zhichao; Lu, Jiangquan; Yang, Chaoyong James

2012-10-21

Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100,000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.

Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy

PubMed Central

Young, Jonathan W; Locke, James C W; Altinok, Alphan; Rosenfeld, Nitzan; Bacarian, Tigran; Swain, Peter S; Mjolsness, Eric; Elowitz, Michael B

2014-01-01

Quantitative single-cell time-lapse microscopy is a powerful method for analyzing gene circuit dynamics and heterogeneous cell behavior. We describe the application of this method to imaging bacteria by using an automated microscopy system. This protocol has been used to analyze sporulation and competence differentiation in Bacillus subtilis, and to quantify gene regulation and its fluctuations in individual Escherichia coli cells. The protocol involves seeding and growing bacteria on small agarose pads and imaging the resulting microcolonies. Images are then reviewed and analyzed using our laboratory's custom MATLAB analysis code, which segments and tracks cells in a frame-to-frame method. This process yields quantitative expression data on cell lineages, which can illustrate dynamic expression profiles and facilitate mathematical models of gene circuits. With fast-growing bacteria, such as E. coli or B. subtilis, image acquisition can be completed in 1 d, with an additional 1–2 d for progressing through the analysis procedure. PMID:22179594

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.

The quantitative and condition-dependent Escherichia coli proteome

PubMed Central

Schmidt, Alexander; Kochanowski, Karl; Vedelaar, Silke; Ahrné, Erik; Volkmer, Benjamin; Callipo, Luciano; Knoops, Kèvin; Bauer, Manuel; Aebersold, Ruedi; Heinemann, Matthias

2016-01-01

Measuring precise concentrations of proteins can provide insights into biological processes. Here, we use efficient protein extraction and sample fractionation and state-of-the-art quantitative mass spectrometry techniques to generate a comprehensive, condition-dependent protein abundance map of Escherichia coli. We measure cellular protein concentrations for 55% of predicted E. coli genes (>2300 proteins) under 22 different experimental conditions and identify methylation and N-terminal protein acetylations previously not known to be prevalent in bacteria. We uncover system-wide proteome allocation, expression regulation, and post-translational adaptations. These data provide a valuable resource for the systems biology and broader E. coli research communities. PMID:26641532

Bacterial pathogen gene abundance and relation to recreational water quality at seven Great Lakes beaches

USGS Publications Warehouse

Oster, Ryan J.; Wijesinghe, Rasanthi U.; Fogarty, Lisa Reynolds; Haack, Sheridan K.; Fogarty, Lisa R.; Tucker, Taaja R.; Riley, Stephen

2014-01-01

Quantitative assessment of bacterial pathogens, their geographic variability, and distribution in various matrices at Great Lakes beaches are limited. Quantitative PCR (qPCR) was used to test for genes from E. coli O157:H7 (eaeO157), shiga-toxin producing E. coli (stx2), Campylobacter jejuni (mapA), Shigella spp. (ipaH), and a Salmonella enterica-specific (SE) DNA sequence at seven Great Lakes beaches, in algae, water, and sediment. Overall, detection frequencies were mapA>stx2>ipaH>SE>eaeO157. Results were highly variable among beaches and matrices; some correlations with environmental conditions were observed for mapA, stx2, and ipaH detections. Beach seasonal mean mapA abundance in water was correlated with beach seasonal mean log10E. coli concentration. At one beach, stx2 gene abundance was positively correlated with concurrent daily E. coli concentrations. Concentration distributions for stx2, ipaH, and mapA within algae, sediment, and water were statistically different (Non-Detect and Data Analysis in R). Assuming 10, 50, or 100% of gene copies represented viable and presumably infective cells, a quantitative microbial risk assessment tool developed by Michigan State University indicated a moderate probability of illness for Campylobacter jejuni at the study beaches, especially where recreational water quality criteria were exceeded. Pathogen gene quantification may be useful for beach water quality management.

Quantitative assessment of Naegleria fowleri and Escherichia coli concentrations within a Texas reservoir.

PubMed

Painter, Stephanie M; Pfau, Russell S; Brady, Jeff A; McFarland, Anne M S

2013-06-01

Previous presence/absence studies have indicated a correlation between the presence of the pathogenic amoeba Naegleria fowleri and the presence of bacteria, such as the fecal indicator Escherichia coli, in environmental surface waters. The objective of this study was to use quantitative real-time polymerase chain reaction (qPCR) methodologies to measure N. fowleri and E. coli concentrations within a Texas reservoir in late summer, and to determine if concentrations of N. fowleri and E. coli were statistically correlated. N. fowleri was detected in water samples from 67% of the reservoir sites tested, with concentrations ranging up to an estimated 26 CE (cell equivalents)/100 mL. E. coli was detected in water samples from 60% of the reservoir sites tested, with concentrations ranging up to 427 CE/100 mL. In this study, E. coli concentrations were not indicative of N. fowleri concentrations.

Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

EPA Science Inventory

Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

Combining Raman and FT-IR spectroscopy with quantitative isotopic labeling for differentiation of E. coli cells at community and single cell levels.

PubMed

Muhamadali, Howbeer; Chisanga, Malama; Subaihi, Abdu; Goodacre, Royston

2015-04-21

There is no doubt that the contribution of microbially mediated bioprocesses toward maintenance of life on earth is vital. However, understanding these microbes in situ is currently a bottleneck, as most methods require culturing these microorganisms to suitable biomass levels so that their phenotype can be measured. The development of new culture-independent strategies such as stable isotope probing (SIP) coupled with molecular biology has been a breakthrough toward linking gene to function, while circumventing in vitro culturing. In this study, for the first time we have combined Raman spectroscopy and Fourier transform infrared (FT-IR) spectroscopy, as metabolic fingerprinting approaches, with SIP to demonstrate the quantitative labeling and differentiation of Escherichia coli cells. E. coli cells were grown in minimal medium with fixed final concentrations of carbon and nitrogen supply, but with different ratios and combinations of (13)C/(12)C glucose and (15)N/(14)N ammonium chloride, as the sole carbon and nitrogen sources, respectively. The cells were collected at stationary phase and examined by Raman and FT-IR spectroscopies. The multivariate analysis investigation of FT-IR and Raman data illustrated unique clustering patterns resulting from specific spectral shifts upon the incorporation of different isotopes, which were directly correlated with the ratio of the isotopically labeled content of the medium. Multivariate analysis results of single-cell Raman spectra followed the same trend, exhibiting a separation between E. coli cells labeled with different isotopes and multiple isotope levels of C and N.

Putative regulatory sites unraveled by network-embedded thermodynamic analysis of metabolome data

PubMed Central

Kümmel, Anne; Panke, Sven; Heinemann, Matthias

2006-01-01

As one of the most recent members of the omics family, large-scale quantitative metabolomics data are currently complementing our systems biology data pool and offer the chance to integrate the metabolite level into the functional analysis of cellular networks. Network-embedded thermodynamic analysis (NET analysis) is presented as a framework for mechanistic and model-based analysis of these data. By coupling the data to an operating metabolic network via the second law of thermodynamics and the metabolites' Gibbs energies of formation, NET analysis allows inferring functional principles from quantitative metabolite data; for example it identifies reactions that are subject to active allosteric or genetic regulation as exemplified with quantitative metabolite data from Escherichia coli and Saccharomyces cerevisiae. Moreover, the optimization framework of NET analysis was demonstrated to be a valuable tool to systematically investigate data sets for consistency, for the extension of sub-omic metabolome data sets and for resolving intracompartmental concentrations from cell-averaged metabolome data. Without requiring any kind of kinetic modeling, NET analysis represents a perfectly scalable and unbiased approach to uncover insights from quantitative metabolome data. PMID:16788595

Effects of human milk fortifier with iron on the bacteriostatic properties of breast milk.

PubMed

Campos, Leticia Fuganti; Repka, João Carlos Domingues; Falcão, Mário Cícero

2013-01-01

To compare bacterial growth in pure colostrum versus colostrum with human milk fortifier (HMF) containing iron. The growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa in 78 samples of pure colostrum or colostrum with added iron-containing HMF was compared. For qualitative analysis, filter paper discs were immersed in samples from each group and incubated for 48 hours with 10(1) colony forming units (CFUs)/mL of each strain. For quantitative assessment, 1 mL of each strain containing 10(7) CFUs/mL was homogenized with 1 mL of either colostrum or colostrum with human milk fortifier, seeded into a Petri dish, and incubated at 37°C. Twenty-four hours later, the number of CFUs was counted. The qualitative analysis showed no difference in bacterial growth. In the quantitative evaluation, E. coli growth in the control group was 29.4±9.7×10(6)CFU/mL, while in the HMF group it was 31.2±10.8×10(6)CFU/mL. The difference between the average growth was 1.9±4.9×10(6)CFU/mL (p=0.001). There were no differences in S. aureus and P. aeruginosa growth. Addition of iron at this concentration reduces breast milk bacteriostatic action against E. coli. Copyright © 2013 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

SSBD: a database of quantitative data of spatiotemporal dynamics of biological phenomena

PubMed Central

Tohsato, Yukako; Ho, Kenneth H. L.; Kyoda, Koji; Onami, Shuichi

2016-01-01

Motivation: Rapid advances in live-cell imaging analysis and mathematical modeling have produced a large amount of quantitative data on spatiotemporal dynamics of biological objects ranging from molecules to organisms. There is now a crucial need to bring these large amounts of quantitative biological dynamics data together centrally in a coherent and systematic manner. This will facilitate the reuse of this data for further analysis. Results: We have developed the Systems Science of Biological Dynamics database (SSBD) to store and share quantitative biological dynamics data. SSBD currently provides 311 sets of quantitative data for single molecules, nuclei and whole organisms in a wide variety of model organisms from Escherichia coli to Mus musculus. The data are provided in Biological Dynamics Markup Language format and also through a REST API. In addition, SSBD provides 188 sets of time-lapse microscopy images from which the quantitative data were obtained and software tools for data visualization and analysis. Availability and Implementation: SSBD is accessible at http://ssbd.qbic.riken.jp. Contact: sonami@riken.jp PMID:27412095

SSBD: a database of quantitative data of spatiotemporal dynamics of biological phenomena.

PubMed

Tohsato, Yukako; Ho, Kenneth H L; Kyoda, Koji; Onami, Shuichi

2016-11-15

Rapid advances in live-cell imaging analysis and mathematical modeling have produced a large amount of quantitative data on spatiotemporal dynamics of biological objects ranging from molecules to organisms. There is now a crucial need to bring these large amounts of quantitative biological dynamics data together centrally in a coherent and systematic manner. This will facilitate the reuse of this data for further analysis. We have developed the Systems Science of Biological Dynamics database (SSBD) to store and share quantitative biological dynamics data. SSBD currently provides 311 sets of quantitative data for single molecules, nuclei and whole organisms in a wide variety of model organisms from Escherichia coli to Mus musculus The data are provided in Biological Dynamics Markup Language format and also through a REST API. In addition, SSBD provides 188 sets of time-lapse microscopy images from which the quantitative data were obtained and software tools for data visualization and analysis. SSBD is accessible at http://ssbd.qbic.riken.jp CONTACT: sonami@riken.jp. © The Author 2016. Published by Oxford University Press.

Quantitative protein expression and cell surface characteristics of Escherichia coli MG1655 biofilms.

PubMed

Mukherjee, Joy; Ow, Saw Yen; Noirel, Josselin; Biggs, Catherine A

2011-02-01

Cell surface physicochemical characterization techniques were combined with quantitative changes in protein expression, to investigate the biological and biophysical changes of Escherichia coli MG1655 cells when grown as a biofilm (BIO). The overall surface charge of BIO cells was found to be less negative, highlighting the need for a lower electrophoretic mobility for attachment to occur. Comparison of the chemical functional groups on the cell surface showed similar profiles, with the absorbance intensity higher for proteins and carbohydrates in the BIO cells. Quantitative proteomic analysis demonstrated that 3 proteins were significantly increased, and 9 proteins significantly decreased in abundance, in cells grown as a BIO compared to their planktonic counterparts, with 7 of these total 12 proteins unique to this study. Proteins showing significant increased or decreased abundance include proteins involved in acid resistance, DNA protection and binding and ABC transporters. Further predictive analysis of the metabolic pathways showed an increased abundance of the amino acid metabolism and tricarboxylic acid (TCA) cycle, with a decrease in expression within the pentose phosphate and glycolysis pathways. It is therefore hypothesized that cells grown as a BIO are still energetically viable potentially using amino acids as an indirect carbon backbone source into the TCA cycle. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of a quantitative H2S MPN test for fecal microbes analysis of water using biochemical and molecular identification.

PubMed

McMahan, Lanakila; Grunden, Amy M; Devine, Anthony A; Sobsey, Mark D

2012-04-15

The sensitivity and specificity of the H(2)S test to detect fecal bacteria in water has been variable and uncertain in previous studies, partly due to its presence-absence results. Furthermore, in groundwater samples false-positive results have been reported, with H(2)S-positive samples containing no fecal coliforms or Escherichia coli. False-negative results also have been reported in other studies, with H(2)S-negative samples found to contain E. coli. Using biochemical and molecular methods and a novel quantitative test format, this research identified the types and numbers of microbial community members present in natural water samples, including fecal indicators and pathogens as well as other bacteria. Representative water sources tested in this study included cistern rainwater, a protected lake, and wells in agricultural and forest settings. Samples from quantitative H(2)S tests of water were further cultured for fecal bacteria by spread plating onto the selective media for detection and isolation of Aeromonas spp., E. coli, Clostridium spp., H(2)S-producers, and species of Salmonella and Shigella. Isolates were then tested for H(2)S production, and identified to the genus and species level using biochemical methods. Terminal Restriction Fragment Length Polymorphisms (TRFLP) was the molecular method employed to quantitatively characterize microbial community diversity. Overall, it was shown that water samples testing positive for H(2)S bacteria also had bacteria of likely fecal origin and waters containing fecal pathogens also were positive for H(2)S bacteria. Of the microorganisms isolated from natural water, greater than 70 percent were identified using TRFLP analysis to reveal a relatively stable group of organisms whose community composition differed with water source and over time. These results further document the validity of the H(2)S test for detecting and quantifying fecal contamination of water. Copyright © 2011 Elsevier Ltd. All rights reserved.

Quantitative assessment of Naegleria fowleri and fecal indicator bacteria in brackish water of Lake Pontchartrain, Louisiana

NASA Astrophysics Data System (ADS)

Xue, J.; Sherchan, S. P.; Lamar, F. G.; Lin, S.; Lamori, J. G.

2017-12-01

Brackish water samples from Lake Pontchartrain in Louisiana were assessed for the presence of pathogenic amoeba Naegleria fowleri, which causes primary amoebic meningoencephalitis (PAM). In our study, quantitative polymerase chain reaction (qPCR) methods were used to determine N. fowleri, E. coli, and Enterococcus in water collected from Lake Pontchartrain. A total of 158 water samples were analyzed over the 10- month sampling period. Statistically significant positive correlation between water temperature and N. fowleri concentration was observed. N. fowleri target sequence was detected at 35.4% (56/158) of the water samples from ten sites around the Lake ranged from 11.6 GC/100 ml water to 457.8 GC/100 ml water. A single factor (ANOVA) analysis shows the average concentration of N. fowleri in summer (119.8 GC/100 ml) was significantly higher than in winter (58.6 GC/100 ml) (p < 0.01). Statistically significant positive correlations were found between N. fowleri and qPCR E. coli results and N. fowleri and colilert E. coli (culture method), respectively. A weak positive correlation between E. coli and Enterococcus was observed from both qPCR (r = 0.27, p < 0.05) and culture based method (r = 0.52, p < 0.05). Meanwhile, significant positive correlation between qPCR and culture based methods for E. coli (r = 0.30, p < 0.05) and Enterococcus concentration was observed (r = 0.26, p < 0.05), respectively. Future research is needed to determine whether sediment is a source of N. fowleri found in the water column.

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

PubMed

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

2015-10-01

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

Time-series analysis of the transcriptome and proteome of Escherichia coli upon glucose repression.

PubMed

Borirak, Orawan; Rolfe, Matthew D; de Koning, Leo J; Hoefsloot, Huub C J; Bekker, Martijn; Dekker, Henk L; Roseboom, Winfried; Green, Jeffrey; de Koster, Chris G; Hellingwerf, Klaas J

2015-10-01

Time-series transcript- and protein-profiles were measured upon initiation of carbon catabolite repression in Escherichia coli, in order to investigate the extent of post-transcriptional control in this prototypical response. A glucose-limited chemostat culture was used as the CCR-free reference condition. Stopping the pump and simultaneously adding a pulse of glucose, that saturated the cells for at least 1h, was used to initiate the glucose response. Samples were collected and subjected to quantitative time-series analysis of both the transcriptome (using microarray analysis) and the proteome (through a combination of 15N-metabolic labeling and mass spectrometry). Changes in the transcriptome and corresponding proteome were analyzed using statistical procedures designed specifically for time-series data. By comparison of the two sets of data, a total of 96 genes were identified that are post-transcriptionally regulated. This gene list provides candidates for future in-depth investigation of the molecular mechanisms involved in post-transcriptional regulation during carbon catabolite repression in E. coli, like the involvement of small RNAs. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

A novel CMOS image sensor system for quantitative loop-mediated isothermal amplification assays to detect food-borne pathogens.

PubMed

Wang, Tiantian; Kim, Sanghyo; An, Jeong Ho

2017-02-01

Loop-mediated isothermal amplification (LAMP) is considered as one of the alternatives to the conventional PCR and it is an inexpensive portable diagnostic system with minimal power consumption. The present work describes the application of LAMP in real-time photon detection and quantitative analysis of nucleic acids integrated with a disposable complementary-metal-oxide semiconductor (CMOS) image sensor. This novel system works as an amplification-coupled detection platform, relying on a CMOS image sensor, with the aid of a computerized circuitry controller for the temperature and light sources. The CMOS image sensor captures the light which is passing through the sensor surface and converts into digital units using an analog-to-digital converter (ADC). This new system monitors the real-time photon variation, caused by the color changes during amplification. Escherichia coli O157 was used as a proof-of-concept target for quantitative analysis, and compared with the results for Staphylococcus aureus and Salmonella enterica to confirm the efficiency of the system. The system detected various DNA concentrations of E. coli O157 in a short time (45min), with a detection limit of 10fg/μL. The low-cost, simple, and compact design, with low power consumption, represents a significant advance in the development of a portable, sensitive, user-friendly, real-time, and quantitative analytic tools for point-of-care diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Quantitative microbial risk assessment for Escherichia coli O157:H7, salmonella, and Listeria monocytogenes in leafy green vegetables consumed at salad bars.

PubMed

Franz, E; Tromp, S O; Rijgersberg, H; van der Fels-Klerx, H J

2010-02-01

Fresh vegetables are increasingly recognized as a source of foodborne outbreaks in many parts of the world. The purpose of this study was to conduct a quantitative microbial risk assessment for Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes infection from consumption of leafy green vegetables in salad from salad bars in The Netherlands. Pathogen growth was modeled in Aladin (Agro Logistics Analysis and Design Instrument) using time-temperature profiles in the chilled supply chain and one particular restaurant with a salad bar. A second-order Monte Carlo risk assessment model was constructed (using @Risk) to estimate the public health effects. The temperature in the studied cold chain was well controlled below 5 degrees C. Growth of E. coli O157:H7 and Salmonella was minimal (17 and 15%, respectively). Growth of L. monocytogenes was considerably greater (194%). Based on first-order Monte Carlo simulations, the average number of cases per year in The Netherlands associated the consumption leafy greens in salads from salad bars was 166, 187, and 0.3 for E. coli O157:H7, Salmonella, and L. monocytogenes, respectively. The ranges of the average number of annual cases as estimated by second-order Monte Carlo simulation (with prevalence and number of visitors as uncertain variables) were 42 to 551 for E. coli O157:H7, 81 to 281 for Salmonella, and 0.1 to 0.9 for L. monocytogenes. This study included an integration of modeling pathogen growth in the supply chain of fresh leafy vegetables destined for restaurant salad bars using software designed to model and design logistics and modeling the public health effects using probabilistic risk assessment software.

Rapid identification and quantification of Campylobacter coli and Campylobacter jejuni by real-time PCR in pure cultures and in complex samples

PubMed Central

2011-01-01

Background Campylobacter spp., especially Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), are recognized as the leading human foodborne pathogens in developed countries. Livestock animals carrying Campylobacter pose an important risk for human contamination. Pigs are known to be frequently colonized with Campylobacter, especially C. coli, and to excrete high numbers of this pathogen in their faeces. Molecular tools, notably real-time PCR, provide an effective, rapid, and sensitive alternative to culture-based methods for the detection of C. coli and C. jejuni in various substrates. In order to serve as a diagnostic tool supporting Campylobacter epidemiology, we developed a quantitative real-time PCR method for species-specific detection and quantification of C. coli and C. jejuni directly in faecal, feed, and environmental samples. Results With a sensitivity of 10 genome copies and a linear range of seven to eight orders of magnitude, the C. coli and C. jejuni real-time PCR assays allowed a precise quantification of purified DNA from C. coli and C. jejuni. The assays were highly specific and showed a 6-log-linear dynamic range of quantification with a quantitative detection limit of approximately 2.5 × 102 CFU/g of faeces, 1.3 × 102 CFU/g of feed, and 1.0 × 103 CFU/m2 for the environmental samples. Compared to the results obtained by culture, both C. coli and C. jejuni real-time PCR assays exhibited a specificity of 96.2% with a kappa of 0.94 and 0.89 respectively. For faecal samples of experimentally infected pigs, the coefficients of correlation between the C. coli or C. jejuni real-time PCR assay and culture enumeration were R2 = 0.90 and R2 = 0.93 respectively. Conclusion The C. coli and C. jejuni real-time quantitative PCR assays developed in this study provide a method capable of directly detecting and quantifying C. coli and C. jejuni in faeces, feed, and environmental samples. These assays represent a new diagnostic tool for studying the epidemiology of Campylobacter by, for instance, investigating the carriage and excretion of C. coli and C. jejuni by pigs from conventional herds. PMID:21600037

Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker.

PubMed

He, Dan; Xie, Xiao; Yang, Fan; Zhang, Heng; Su, Haomiao; Ge, Yun; Song, Haiping; Chen, Peng R

2017-11-13

A genetically encoded, multifunctional photocrosslinker was developed for quantitative and comparative proteomics. By bearing a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead, this probe enables the enrichment of transient and low-abundance prey proteins after intracellular photocrosslinking and prey-bait separation, which can be subject to stable isotope dimethyl labeling and mass spectrometry analysis. This quantitative strategy (termed isoCAPP) allowed a comparative proteomic approach to be adopted to identify the proteolytic substrates of an E. coli protease-chaperone dual machinery DegP. Two newly identified substrates were subsequently confirmed by proteolysis experiments. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed Central

Elkhatib, Walid F.

2016-01-01

The purpose of this study was to: (i) evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram) against Escherichia coli; (ii) investigate the effects of these honeys on bacterial ultrastructure; and (iii) assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism. The minimum inhibitory concentration (MIC) of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR) analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed “ghost” cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets. PMID:26954570

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

PubMed

Wasfi, Reham; Elkhatib, Walid F; Khairalla, Ahmed S

2016-01-01

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

The Influences of LuxX in "Escherichia Coli" Biofilm Formation and Improving Teacher Quality through the Bio-Bus Program

ERIC Educational Resources Information Center

Robbins, Chandan Morris

2012-01-01

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…

In vitro adherence of radioactively labeled Escherichia coli in normal and cystitis-prone females

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

Parsons, C.L.; Anwar, H.; Stauffer, C.

Numerous investigators report data obtained using an in vitro quantitative assay for measuring bacterial adherence to epithelial cells. In the modified assay described here, we eliminated the need for visual counting of bacteria by incorporating the use of radioactively labeled Escherichia coli. This allowed quantitation of bacterial adherence to as many as 50,000 vaginal cells, whereas the visual counting system limits the determination to perhaps 50 cells. Using the modified method, we found no statistically significant differences among values for adherence of E. coli type 04 to the vaginal cells of control and cystitis-prone women at either pH 6.4 ormore » 4.0.« less

Quantitative risk assessment of E. coli in street-vended cassava-based delicacies in the Philippines

NASA Astrophysics Data System (ADS)

Mesias, I. C. P.

2018-01-01

In the Philippines, rootcrop-based food products are gaining popularity in street food trade. However, a number of street-vended food products in the country are reported to be contaminated with E. coli posing possible risk among consumers. In this study, information on quantitative risk assessment of E. coli in street-vended cassava-based delicacies was generated. The assessment started with the prevalence and concentration of E. coli at post production in packages of the cassava-based delicacies. Combase growth predictor was used to trace the microbial population of E. coli in each step of the food chain. The @Risk software package, version 6 (Palisade USA) was used to run the simulations. Scenarios in the post-production to consumption pathway were simulated. The effect was then assessed in relation to exposure to the defined infective dose. In the worst case scenario, a minimum and most likely concentration of 6.3 and 7.8 log CFU of E. coli per serving respectively were observed. The simulation revealed that lowering the temperature in the chain considerably decreased the E. coli concentration prior to consumption and subsequently decreased the percentage of exposure to the infective dose. Exposure to infective dose however was increased with longer lag time from postproduction to consumption.

Comparison of the quantitative dry culture methods with both conventional media and most probable number method for the enumeration of coliforms and Escherichia coli/coliforms in food.

PubMed

Teramura, H; Sota, K; Iwasaki, M; Ogihara, H

2017-07-01

Sanita-kun™ CC (coliform count) and EC (Escherichia coli/coliform count), sheet quantitative culture systems which can avoid chromogenic interference by lactase in food, were evaluated in comparison with conventional methods for these bacteria. Based on the results of inclusivity and exclusivity studies using 77 micro-organisms, sensitivity and specificity of both Sanita-kun™ met the criteria for ISO 16140. Both media were compared with deoxycholate agar, violet red bile agar, Merck Chromocult™ coliform agar (CCA), 3M Petrifilm™ CC and EC (PEC) and 3-tube MPN, as reference methods, in 100 naturally contaminated food samples. The correlation coefficients of both Sanita-kun™ for coliform detection were more than 0·95 for all comparisons. For E. coli detection, Sanita-kun™ EC was compared with CCA, PEC and MPN in 100 artificially contaminated food samples. The correlation coefficients for E. coli detection of Sanita-kun™ EC were more than 0·95 for all comparisons. There were no significant differences in all comparisons when conducting a one-way analysis of variance (anova). Both Sanita-kun™ significantly inhibited colour interference by lactase when inhibition of enzymatic staining was assessed using 40 natural cheese samples spiked with coliform. Our results demonstrated Sanita-kun™ CC and EC are suitable alternatives for the enumeration of coliforms and E. coli/coliforms, respectively, in a variety of foods, and specifically in fermented foods. Current chromogenic media for coliforms and Escherichia coli/coliforms have enzymatic coloration due to breaking down of chromogenic substrates by food lactase. The novel sheet culture media which have film layer to avoid coloration by food lactase have been developed for enumeration of coliforms and E. coli/coliforms respectively. In this study, we demonstrated these media had comparable performance with reference methods and less interference by food lactase. These media have a possibility not only to be useful alternatives but also to contribute for accurate enumeration of these bacteria in a variety of foods, and specifically in fermented foods. © 2017 The Society for Applied Microbiology.

A metabolomics-based method for studying the effect of yfcC gene in Escherichia coli on metabolism.

PubMed

Wang, Xiyue; Xie, Yuping; Gao, Peng; Zhang, Sufang; Tan, Haidong; Yang, Fengxu; Lian, Rongwei; Tian, Jing; Xu, Guowang

2014-04-15

Metabolomics is a potent tool to assist in identifying the function of unknown genes through analysis of metabolite changes in the context of varied genetic backgrounds. However, the availability of a universal unbiased profiling analysis is still a big challenge. In this study, we report an optimized metabolic profiling method based on gas chromatography-mass spectrometry for Escherichia coli. It was found that physiological saline at -80°C could ensure satisfied metabolic quenching with less metabolite leakage. A solution of methanol/water (21:79, v/v) was proved to be efficient for intracellular metabolite extraction. This method was applied to investigate the metabolome difference among wild-type E. coli, its yfcC deletion, and overexpression mutants. Statistical and bioinformatic analysis of the metabolic profiling data indicated that the expression of yfcC potentially affected the metabolism of glyoxylate shunt. This finding was further validated by real-time quantitative polymerase chain reactions showing that expression of aceA and aceB, the key genes in glyoxylate shunt, was upregulated by yfcC. This study exemplifies the robustness of the proposed metabolic profiling analysis strategy and its potential roles in investigating unknown gene functions in view of metabolome difference. Copyright © 2014 Elsevier Inc. All rights reserved.

Quantitative differences in adhesiveness of type 1 fimbriated Escherichia coli due to structural differences in fimH genes.

PubMed Central

Sokurenko, E V; Courtney, H S; Maslow, J; Siitonen, A; Hasty, D L

1995-01-01

Type 1 fimbriae are heteropolymeric surface organelles responsible for the D-mannose-sensitive (MS) adhesion of Escherichia coli. We recently reported that variation of receptor specificity of type 1 fimbriae can result solely from minor alterations in the structure of the gene for the FimH adhesin subunit. To further study the relationship between allelic variation of the fimH gene and adhesive properties of type 1 fimbriae, the fimH genes from five additional strains were cloned and used to complement the FimH deletion in E. coli KB18. When the parental and recombinant strains were tested for adhesion to immobilized mannan, a wide quantitative range in the ability of bacteria to adhere was noted. The differences in adhesion do not appear to be due to differences in the levels of fimbriation or relative levels of incorporation of FimH, because these parameters were similar in low-adhesion and high-adhesion strains. The nucleotide sequence for each of the fimH genes was determined. Analysis of deduced FimH sequences allowed identification of two sequence homology groups, based on the presence of Asn-70 and Ser-78 or Ser-70 and Asn-78 residues. The consensus sequences for each group conferred very low adhesion activity, and this low-adhesion phenotype predominated among a group of 43 fecal isolates. Strains isolated from a different host niche, the urinary tract, expressed type 1 fimbriae that conferred an increased level of adhesion. The results presented here strongly suggest that the quantitative variations in MS adhesion are due primarily to structural differences in the FimH adhesin. The observed differences in MS adhesion among populations of E. coli isolated from different host niches call attention to the possibility that phenotypic variants of FimH may play a functional role in populations dynamics. PMID:7601831

Development of an Escherichia coli K12-specific quantitative polymerase chain reaction assay and DNA isolation suited to biofilms associated with iron drinking water pipe corrosion products

EPA Science Inventory

Escherichia coli is one of the most commonly used fecal indicator organisms for drinking water and groundwater systems. In order to understand various biogeochemical and biophysical factors affecting its interactions with biofilms, E. coli K12 was chosen as a model organism. A Ta...

Development of an Escherichia coli K12-specific quantitative polymerase chain reaction assay and DNA isolation suited to biofilms associated with iron drinking water pipe corrosion products.

PubMed

Lu, Jingrang; Gerke, Tammie L; Buse, Helen Y; Ashbolt, Nicholas J

2014-12-01

A quantitative polymerase chain reaction assay (115 bp amplicon) specific to Escherichia coli K12 with an ABI(TM) internal control was developed based on sequence data encoding the rfb gene cluster. Assay specificity was evaluated using three E. coli K12 strains (ATCC W3110, MG1655 & DH1), 24 non-K12 E. coli and 23 bacterial genera. The biofilm detection limit was 10(3) colony-forming units (CFU) E. coli K12 mL(-1), but required a modified protocol, which included a bio-blocker Pseudomonas aeruginosa with ethylenediaminetetraacetic acid buffered to pH 5 prior to cell lysis/DNA extraction. The novel protocol yielded the same sensitivity for drinking water biofilms associated with Fe3O4 (magnetite)-coated SiO2 (quartz) grains and biofilm-surface iron corrosion products from a drinking water distribution system. The novel DNA extraction protocol and specific E. coli K12 assay are sensitive and robust enough for detection and quantification within iron drinking water pipe biofilms, and are particularly well suited for studying enteric bacterial interactions within biofilms.

Successful Validation of RNA Purification and Quantitative Real-Time PCR Analysis of Gene Expression on the International Space Station

NASA Technical Reports Server (NTRS)

Tran, L.; Parra, Macarena P.; Jung, J.; Boone, T.; Schonfeld, Julie; Almeida, Eduardo

2017-01-01

The NASA Ames WetLab-2 system was developed to offer new on-orbit gene expression analysis capabilities to ISS researchers and can be used to conduct on-orbit RNA isolation and quantitative real time PCR (RT-qPCR) analysis of gene expression from a wide range of biological samples ranging from microbes to mammalian tissues. On orbit validation included three quantitative PCR (qPCR) runs using an E. coli genomic DNA template pre-loaded at three different concentrations. The flight Ct values for the DNA standards showed no statistically significant differences relative to ground controls although there was increased noise in Ct curves, likely due to microgravity-related bubble retention in the optical windows. RNA was successfully purified from both E. coli and mouse liver samples and successfully generated singleplex, duplex and triplex data although with higher standard deviations than ground controls, also likely due to bubbles. Using volunteer science activities, a potential bubble reduction strategy was tested and resulted in smooth amplification curves and tighter Cts between replicates. The WetLab-2 validation experiment demonstrates a novel molecular biology workbench on ISS which allows scientists to purify and stabilize RNA, and to conduct RT-qPCR analyses on-orbit with rapid results. This novel ability is an important step towards utilizing ISS as a National Laboratory facility with the capability to conduct and adjust science experiments in real time without sample return, and opens new possibilities for rapid medical diagnostics and biological environmental monitoring on ISS.

Hierarchical Flowerlike Gold Nanoparticles Labeled Immunochromatography Test Strip for Highly Sensitive Detection of Escherichia coli O157:H7.

PubMed

Zhang, Lei; Huang, Youju; Wang, Jingyun; Rong, Yun; Lai, Weihua; Zhang, Jiawei; Chen, Tao

2015-05-19

Gold nanoparticles (AuNPs) labeled lateral-flow test strip immunoassay (LFTS) has been widely used in biomedical, feed/food, and environmental analysis fields. Conventional ILFS assay usually uses spherical AuNPs as labeled probes and shows low detection sensitivity, which further limits its widespread practical application. Unlike spherical AuNP used as labeled probe in conventional ILFS, in our present study, a hierarchical flowerlike AuNP specific probe was designed for LFTS and further used to detect Escherichia coli O157:H7 (E. coli O157:H7). Three types of hierarchical flowerlike AuNPs, such as tipped flowerlike, popcornlike, and large-sized flowerlike AuNPs were synthesized in a one-step method. Compared with other two kinds of Au particles, tipped flowerlike AuNPs probes for LFTS particularly exhibited highly sensitive detection of E. coli O157:H7. The remarkable improvement of detection sensitivity of tipped flowerlike AuNPs probes can be achieved even as low as 10(3) colony-forming units (CFU)/mL by taking advantages of its appropriate size and hierarchical structures, which is superior over the detection performance of conventional LFTS. Using this novel tipped flower AuNPs probes, quantitative detection of E. coli O157:H7 can be obtained partially in a wide concentration range with good repeatability. This hierarchical tipped flower-shaped AuNPs probe for LFTS is promising for the practical applications in widespread analysis fields.

Quantitative Metabolomics Reveals an Epigenetic Blueprint for Iron Acquisition in Uropathogenic Escherichia coli

PubMed Central

Henderson, Jeffrey P.; Crowley, Jan R.; Pinkner, Jerome S.; Walker, Jennifer N.; Tsukayama, Pablo; Stamm, Walter E.; Hooton, Thomas M.; Hultgren, Scott J.

2009-01-01

Bacterial pathogens are frequently distinguished by the presence of acquired genes associated with iron acquisition. The presence of specific siderophore receptor genes, however, does not reliably predict activity of the complex protein assemblies involved in synthesis and transport of these secondary metabolites. Here, we have developed a novel quantitative metabolomic approach based on stable isotope dilution to compare the complement of siderophores produced by Escherichia coli strains associated with intestinal colonization or urinary tract disease. Because uropathogenic E. coli are believed to reside in the gut microbiome prior to infection, we compared siderophore production between urinary and rectal isolates within individual patients with recurrent UTI. While all strains produced enterobactin, strong preferential expression of the siderophores yersiniabactin and salmochelin was observed among urinary strains. Conventional PCR genotyping of siderophore receptors was often insensitive to these differences. A linearized enterobactin siderophore was also identified as a product of strains with an active salmochelin gene cluster. These findings argue that qualitative and quantitative epi-genetic optimization occurs in the E. coli secondary metabolome among human uropathogens. Because the virulence-associated biosynthetic pathways are distinct from those associated with rectal colonization, these results suggest strategies for virulence-targeted therapies. PMID:19229321

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

PubMed

Ahmed, W; Gyawali, P; Toze, S

2015-03-03

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

Quantitative assessment of the microbial risk of leafy greens from farm to consumption: preliminary framework, data, and risk estimates.

PubMed

Danyluk, Michelle D; Schaffner, Donald W

2011-05-01

This project was undertaken to relate what is known about the behavior of Escherichia coli O157:H7 under laboratory conditions and integrate this information to what is known regarding the 2006 E. coli O157:H7 spinach outbreak in the context of a quantitative microbial risk assessment. The risk model explicitly assumes that all contamination arises from exposure in the field. Extracted data, models, and user inputs were entered into an Excel spreadsheet, and the modeling software @RISK was used to perform Monte Carlo simulations. The model predicts that cut leafy greens that are temperature abused will support the growth of E. coli O157:H7, and populations of the organism may increase by as much a 1 log CFU/day under optimal temperature conditions. When the risk model used a starting level of -1 log CFU/g, with 0.1% of incoming servings contaminated, the predicted numbers of cells per serving were within the range of best available estimates of pathogen levels during the outbreak. The model predicts that levels in the field of -1 log CFU/g and 0.1% prevalence could have resulted in an outbreak approximately the size of the 2006 E. coli O157:H7 outbreak. This quantitative microbial risk assessment model represents a preliminary framework that identifies available data and provides initial risk estimates for pathogenic E. coli in leafy greens. Data gaps include retail storage times, correlations between storage time and temperature, determining the importance of E. coli O157:H7 in leafy greens lag time models, and validation of the importance of cross-contamination during the washing process.

Maximum entropy modeling of metabolic networks by constraining growth-rate moments predicts coexistence of phenotypes

NASA Astrophysics Data System (ADS)

De Martino, Daniele

2017-12-01

In this work maximum entropy distributions in the space of steady states of metabolic networks are considered upon constraining the first and second moments of the growth rate. Coexistence of fast and slow phenotypes, with bimodal flux distributions, emerges upon considering control on the average growth (optimization) and its fluctuations (heterogeneity). This is applied to the carbon catabolic core of Escherichia coli where it quantifies the metabolic activity of slow growing phenotypes and it provides a quantitative map with metabolic fluxes, opening the possibility to detect coexistence from flux data. A preliminary analysis on data for E. coli cultures in standard conditions shows degeneracy for the inferred parameters that extend in the coexistence region.

Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product.

PubMed

Fazelahi, Mansoureh; Kia, Vahid; Kaghazian, Hooman; Paryan, Mahdi

2017-11-26

Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli's and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (10 1 to 10 7 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.

Proteomic Profiling of Recombinant Escherichia coli in High-Cell- Density Fermentations for Improved Production of an Antibody Fragment Biopharmaceutical

PubMed Central

Aldor, Ilana S.; Krawitz, Denise C.; Forrest, William; Chen, Christina; Nishihara, Julie C.; Joly, John C.; Champion, Kathleen M.

2005-01-01

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using a t-test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield. PMID:15811994

A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli

PubMed Central

Sashital, Dipali G; Greeman, Candacia A; Lyumkis, Dmitry; Potter, Clinton S; Carragher, Bridget; Williamson, James R

2014-01-01

Ribosome assembly is a complex process involving the folding and processing of ribosomal RNAs (rRNAs), concomitant binding of ribosomal proteins (r-proteins), and participation of numerous accessory cofactors. Here, we use a quantitative mass spectrometry/electron microscopy hybrid approach to determine the r-protein composition and conformation of 30S ribosome assembly intermediates in Escherichia coli. The relative timing of assembly of the 3′ domain and the formation of the central pseudoknot (PK) structure depends on the presence of the assembly factor RimP. The central PK is unstable in the absence of RimP, resulting in the accumulation of intermediates in which the 3′-domain is unanchored and the 5′-domain is depleted for r-proteins S5 and S12 that contact the central PK. Our results reveal the importance of the cofactor RimP in central PK formation, and introduce a broadly applicable method for characterizing macromolecular assembly in cells. DOI: http://dx.doi.org/10.7554/eLife.04491.001 PMID:25313868

Collection methods and quality assessment for Esche-richia coli, water quality, and microbial source tracking data within Tumacácori National Historical Park and the upper Santa Cruz River, Arizona, 2015-16

USGS Publications Warehouse

Paretti, Nicholas; Coes, Alissa L.; Kephart, Christopher M.; Mayo, Justine

2018-03-05

Tumacácori National Historical Park protects the culturally important Mission, San José de Tumacácori, while also managing a portion of the ecologically diverse riparian corridor of the Santa Cruz River. This report describes the methods and quality assurance procedures used in the collection of water samples for the analysis of Escherichia coli (E. coli), microbial source tracking markers, suspended sediment, water-quality parameters, turbidity, and the data collection for discharge and stage; the process for data review and approval is also described. Finally, this report provides a quantitative assessment of the quality of the E. coli, microbial source tracking, and suspended sediment data.The data-quality assessment revealed that bias attributed to field and laboratory contamination was minimal, with E. coli detections in only 3 out of 33 field blank samples analyzed. Concentrations in the field blanks were several orders of magnitude lower than environmental concentrations. The microbial source tracking (MST) field blank was below the detection limit for all MST markers analyzed. Laboratory blanks for E. coli at the USGS Arizona Water Science Center and laboratory blanks for MST markers at the USGS Ohio Water Microbiology Laboratory were all below the detection limit. Irreplicate data for E. coli and suspended sediment indicated that bias was not introduced to the data by combining samples collected using discrete sampling methods with samples collected using automatic sampling methods.The split and sequential E. coli replicate data showed consistent analytical variability and a single equation was developed to explain the variability of E. coli concentrations. An additional analysis of analytical variability for E. coli indicated analytical variability around 18 percent relative standard deviation and no trend was observed in the concentration during the processing and analysis of multiple split-replicates. Two replicate samples were collected for MST and individual markers were compared for a base flow and flood sample. For the markers found in common between the two types of samples, the relative standard deviation for the base flow sample was more than 3 times greater than the markers in the flood sample. Sequential suspended sediment replicates had a relative standard deviation of about 1.3 percent, indicating that environmental and analytical variability was minimal.A holding time review and laboratory study analysis supported the extended holding times required for this investigation. Most concentrations for flood and base-flow samples were within the theoretical variability specified in the most probable number approach suggesting that extended hold times did not overly influence the final concentrations reported.

Predicting Gene Expression Level from Relative Codon Usage Bias: An Application to Escherichia coli Genome

PubMed Central

Roymondal, Uttam; Das, Shibsankar; Sahoo, Satyabrata

2009-01-01

We present an expression measure of a gene, devised to predict the level of gene expression from relative codon bias (RCB). There are a number of measures currently in use that quantify codon usage in genes. Based on the hypothesis that gene expressivity and codon composition is strongly correlated, RCB has been defined to provide an intuitively meaningful measure of an extent of the codon preference in a gene. We outline a simple approach to assess the strength of RCB (RCBS) in genes as a guide to their likely expression levels and illustrate this with an analysis of Escherichia coli (E. coli) genome. Our efforts to quantitatively predict gene expression levels in E. coli met with a high level of success. Surprisingly, we observe a strong correlation between RCBS and protein length indicating natural selection in favour of the shorter genes to be expressed at higher level. The agreement of our result with high protein abundances, microarray data and radioactive data demonstrates that the genomic expression profile available in our method can be applied in a meaningful way to the study of cell physiology and also for more detailed studies of particular genes of interest. PMID:19131380

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.

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

PubMed

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

2017-02-01

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

Quantitative PCR Profiling of Escherichia coli in Livestock Feces Reveals Increased Population Resilience Relative to Culturable Counts under Temperature Extremes.

PubMed

Oliver, David M; Bird, Clare; Burd, Emmy; Wyman, Michael

2016-09-06

The relationship between culturable counts (CFU) and quantitative PCR (qPCR) cell equivalent counts of Escherichia coli in dairy feces exposed to different environmental conditions and temperature extremes was investigated. Fecal samples were collected in summer and winter from dairy cowpats held under two treatments: field-exposed versus polytunnel-protected. A significant correlation in quantified E. coli was recorded between the qPCR and culture-based methods (r = 0.82). Evaluation of the persistence profiles of E. coli over time revealed no significant difference in the E. coli numbers determined as either CFU or gene copies during the summer for the field-exposed cowpats, whereas significantly higher counts were observed by qPCR for the polytunnel-protected cowpats, which were exposed to higher ambient temperatures. In winter, the qPCR returned significantly higher counts of E. coli for the field-exposed cowpats, thus representing a reversal of the findings from the summer sampling campaign. Results from this study suggest that with increasing time post-defecation and with the onset of challenging environmental conditions, such as extremes in temperature, culture-based counts begin to underestimate the true resilience of viable E. coli populations in livestock feces. This is important not only in the long term as the Earth changes in response to climate-change drivers but also in the short term during spells of extremely cold or hot weather.

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

PubMed

Limayem, Alya; Martin, Elizabeth M

2014-01-01

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

Influence of Vacuum Cooling on Escherichia coli O157:H7 Infiltration in Fresh Leafy Greens via a Multiphoton-Imaging Approach

PubMed Central

Vonasek, Erica

2015-01-01

Microbial pathogen infiltration in fresh leafy greens is a significant food safety risk factor. In various postharvest operations, vacuum cooling is a critical process for maintaining the quality of fresh produce. The overall goal of this study was to evaluate the risk of vacuum cooling-induced infiltration of Escherichia coli O157:H7 into lettuce using multiphoton microscopy. Multiphoton imaging was chosen as the method to locate E. coli O157:H7 within an intact lettuce leaf due to its high spatial resolution, low background fluorescence, and near-infrared (NIR) excitation source compared to those of conventional confocal microscopy. The variables vacuum cooling, surface moisture, and leaf side were evaluated in a three-way factorial study with E. coli O157:H7 on lettuce. A total of 188 image stacks were collected. The images were analyzed for E. coli O157:H7 association with stomata and E. coli O157:H7 infiltration. The quantitative imaging data were statistically analyzed using analysis of variance (ANOVA). The results indicate that the low-moisture condition led to an increased risk of microbial association with stomata (P < 0.05). Additionally, the interaction between vacuum cooling levels and moisture levels led to an increased risk of infiltration (P < 0.05). This study also demonstrates the potential of multiphoton imaging for improving sensitivity and resolution of imaging-based measurements of microbial interactions with intact leaf structures, including infiltration. PMID:26475109

Abundance of Naegleria fowleri in roof-harvested rainwater tank samples from two continents.

PubMed

Waso, Monique; Dobrowsky, Penelope Heather; Hamilton, Kerry Ann; Puzon, Geoffrey; Miller, Haylea; Khan, Wesaal; Ahmed, Warish

2018-02-01

Roof-harvested rainwater (RHRW) has been used as an alternative source of water in water scarce regions of many countries. The microbiological and chemical quality of RHRW has been questioned due to the presence of bacterial and protozoan pathogens. However, information on the occurrence of pathogenic amoeba in RHRW tank samples is needed due to their health risk potential and known associations with opportunistic pathogens. Therefore, this study aims to determine the quantitative occurrence of Naegleria fowleri in RHRW tank samples from Southeast Queensland (SEQ), Australia (AU), and the Kleinmond Housing Scheme located in Kleinmond, South Africa (SA). In all, 134 and 80 RHRW tank samples were collected from SEQ, and the Kleinmond Housing Scheme, Western Cape, SA, respectively. Quantitative PCR (qPCR) assays were used to measure the concentrations of N. fowleri, and culture-based methods were used to measure fecal indicator bacteria (FIB) Escherichia coli (E. coli) and Enterococcus spp. Of the 134 tank water samples tested from AU, 69 and 62.7% were positive for E. coli, and Enterococcus spp., respectively. For the SA tank water samples, FIB analysis was conducted for samples SA-T41 to SA-T80 (n = 40). Of the 40 samples analyzed from SA, 95 and 35% were positive for E. coli and Enterococcus spp., respectively. Of the 134 water samples tested in AU, 15 (11.2%) water samples were positive for N. fowleri, and the concentrations ranged from 1.7 × 10 2 to 3.6 × 10 4 gene copies per 100 mL of water. Of the 80 SA tank water samples screened for N. fowleri, 15 (18.8%) tank water samples were positive for N. fowleri and the concentrations ranged from 2.1 × 10 1 to 7.8 × 10 4 gene copies per 100 mL of tank water. The prevalence of N. fowleri in RHRW tank samples from AU and SA thus warrants further development of dose-response models for N. fowleri and a quantitative microbial risk assessment (QMRA) to inform and prioritize strategies for reducing associated public health risks.

Highly sensitive bacterial susceptibility test against penicillin using parylene-matrix chip.

PubMed

Park, Jong-Min; Kim, Jo-Il; Song, Hyun-Woo; Noh, Joo-Yoon; Kang, Min-Jung; Pyun, Jae-Chul

2015-09-15

This work presented a highly sensitive bacterial antibiotic susceptibility test through β-lactamase assay using Parylene-matrix chip. β-lactamases (EC 3.5.2.6) are an important family of enzymes that confer resistance to β-lactam antibiotics by catalyzing the hydrolysis of these antibiotics. Here we present a highly sensitive assay to quantitate β-lactamase-mediated hydrolysis of penicillin into penicilloic acid. Typically, MALDI-TOF mass spectrometry has been used to quantitate low molecular weight analytes and to discriminate them from noise peaks of matrix fragments that occur at low m/z ratios (m/z<500). The β-lactamase assay for the Escherichia coli antibiotic susceptibility test was carried out using Parylene-matrix chip and MALDI-TOF mass spectrometry. The Parylene-matrix chip was successfully used to quantitate penicillin (m/z: [PEN+H](+)=335.1 and [PEN+Na](+)=357.8) and penicilloic acid (m/z: [PA+H](+)=353.1) in a β-lactamase assay with minimal interference of low molecular weight noise peaks. The β-lactamase assay was carried out with an antibiotic-resistant E. coli strain and an antibiotic-susceptible E. coli strain, revealing that the minimum number of E. coli cells required to screen for antibiotic resistance was 1000 cells for the MALDI-TOF mass spectrometry/Parylene-matrix chip assay. Copyright © 2015 Elsevier B.V. All rights reserved.

Gold Nanoparticle Labeling Based ICP-MS Detection/Measurement of Bacteria, and Their Quantitative Photothermal Destruction

PubMed Central

Lin, Yunfeng

2015-01-01

Bacteria such as Salmonella and E. coli present a great challenge in public health care in today’s society. Protection of public safety against bacterial contamination and rapid diagnosis of infection require simple and fast assays for the detection and elimination of bacterial pathogens. After utilizing Salmonella DT104 as an example bacterial strain for our investigation, we report a rapid and sensitive assay for the qualitative and quantitative detection of bacteria by using antibody affinity binding, popcorn shaped gold nanoparticle (GNPOPs) labeling, surfance enchanced Raman spectroscopy (SERS), and inductively coupled plasma mass spectrometry (ICP-MS) detection. For qualitative analysis, our assay can detect Salmonella within 10 min by Raman spectroscopy; for quantitative analysis, our assay has the ability to measure as few as 100 Salmonella DT104 in a 1 mL sample (100 CFU/mL) within 40 min. Based on the quantitative detection, we investigated the quantitative destruction of Salmonella DT104, and the assay’s photothermal efficiency in order to reduce the amount of GNPOPs in the assay to ultimately to eliminate any potential side effects/toxicity to the surrounding cells in vivo. Results suggest that our assay may serve as a promising candidate for qualitative and quantitative detection and elimination of a variety of bacterial pathogens. PMID:26417447

Quantifying translational coupling in E. coli synthetic operons using RBS modulation and fluorescent reporters.

PubMed

Levin-Karp, Ayelet; Barenholz, Uri; Bareia, Tasneem; Dayagi, Michal; Zelcbuch, Lior; Antonovsky, Niv; Noor, Elad; Milo, Ron

2013-06-21

Translational coupling is the interdependence of translation efficiency of neighboring genes encoded within an operon. The degree of coupling may be quantified by measuring how the translation rate of a gene is modulated by the translation rate of its upstream gene. Translational coupling was observed in prokaryotic operons several decades ago, but the quantitative range of modulation translational coupling leads to and the factors governing this modulation were only partially characterized. In this study, we systematically quantify and characterize translational coupling in E. coli synthetic operons using a library of plasmids carrying fluorescent reporter genes that are controlled by a set of different ribosome binding site (RBS) sequences. The downstream gene expression level is found to be enhanced by the upstream gene expression via translational coupling with the enhancement level varying from almost no coupling to over 10-fold depending on the upstream gene's sequence. Additionally, we find that the level of translational coupling in our system is similar between the second and third locations in the operon. The coupling depends on the distance between the stop codon of the upstream gene and the start codon of the downstream gene. This study is the first to systematically and quantitatively characterize translational coupling in a synthetic E. coli operon. Our analysis will be useful in accurate manipulation of gene expression in synthetic biology and serves as a step toward understanding the mechanisms involved in translational expression modulation.

Analytical performance of reciprocal isotope labeling of proteome digests for quantitative proteomics and its application for comparative studies of aerobic and anaerobic Escherichia coli proteomes.

PubMed

Lo, Andy; Weiner, Joel H; Li, Liang

2013-09-17

Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

Electrochemical immunosensor assay (EIA) for sensitive detection of E. coli O157:H7 with signal amplification on a SG-PEDOT-AuNPs electrode interface.

PubMed

Guo, Yuna; Wang, Yu; Liu, Su; Yu, Jinghua; Wang, Hongzhi; Cui, Min; Huang, Jiadong

2015-01-21

A novel electrochemical immunosensor assay (EIA) for highly sensitive and specific detection of Escherichia coli O157:H7 has been developed. This immunosensor is constructed by the assembly of capture antibody on SG-PEDOT-AuNPs composites modified glass carbon electrode. In the presence of target E. coli O157:H7, horseradish peroxidase (HRP)-labeled antibody is captured on the electrode surface to form a sandwich-type system via the specific identification. As a result, E. coli O157:H7 detection is realized by outputting a redox current from electro-reduction of hydrogen peroxide reaction catalyzed by HRP. In our assay, the combination of the unique properties of sulfonated graphene (SG) and gold nanoparticles (AuNPs) can not only accelerate electron transfer on electrode interface, but also provide an excellent scaffold for the conjugation of capture antibody that significantly improves the target capture efficiency and enhances the sensitivity of the biosensor. The results reveal the calibration plot obtained for E. coli O157:H7 is approximately linear from 7.8 × 10-7.8 × 10(6) colony-forming unit (cfu) mL(-1) with the limit of detection of 3.4 × 10 cfu mL(-1). In addition, the biosensor has been successfully applied to the quantitative assay of E. coli O157:H7 in synthetic samples (spring water and milk). Hence, the developed electrochemical-based immunosensor might provide a useful and practical tool for E. coli O157:H7 determination and related food safety analysis and clinical diagnosis.

Quantitative assessment of Naegleria fowleri and fecal indicator bacteria in brackish water of Lake Pontchartrain, Louisiana.

PubMed

Xue, Jia; Lamar, Frederica G; Zhang, Bowen; Lin, Siyu; Lamori, Jennifer G; Sherchan, Samendra P

2018-05-01

Brackish water samples from Lake Pontchartrain in Louisiana were assessed for the presence of pathogenic amoeba Naegleria fowleri, which causes primary amoebic meningoencephalitis (PAM). In our study, quantitative polymerase chain reaction (qPCR) methods were used to determine N. fowleri, E. coli, and enterococci in water collected from Lake Pontchartrain. N. fowleri target sequence was detected in 35.4% (56/158) of the water samples from ten sites around the lake. Statistically significant positive correlations between N. fowleri concentration and water temperature as well as E. coli (qPCR) were observed. Multiple linear regression (MLR) model shows seasonal factor (summer or winter) has significant effect on the concentration of N. fowleri, E. coli and enterococci (qPCR) concentration. Significant positive relationships between E. coli and enterococci was observed from both qPCR (r=0.25) and culture based method (r=0.54). Meanwhile, significant positive correlation between qPCR and culture based methods for enterococci concentration was observed (r=0.33). In our study, water temperature and E. coli concentration were indicative of N. fowleri concentrations in brackish water environment. Future research is needed to determine whether sediment is a source of N. fowleri found in the water column. Copyright © 2017 Elsevier B.V. All rights reserved.

Epidemiology and risk factors for infections due to AmpC β-lactamase-producing Escherichia coli.

PubMed

Pascual, Vanesa; Ortiz, Gabriel; Simó, Maria; Alonso, Noemí; Garcia, Maria Consol; Xercavins, Mariona; Rivera, Alba; Morera, Maria Antonia; Miró, Elisenda; Espejo, Elena; Navarro, Ferran; Gurguí, Mercè; Pérez, Josefa; Rodríguez-Carballeira, Mónica; Garau, Javier; Calbo, Esther

2015-03-01

To describe the prevalence and risk factors for infection due to AmpC β-lactamase-producing Escherichia coli (AmpC-EC). For the prevalence study, all clinical isolates of E. coli with reduced susceptibility to third-generation cephalosporins were prospectively included from June 2010 to November 2011. For risk factor analysis, a case-control study was conducted. Cases were patients with an infection due to AmpC-EC. Controls were patients infected with cephalosporin-susceptible E. coli, matched 1 : 2. Detection of blaAmpC genes was done with a multiplex AmpC-PCR, and hyperproduction of E. coli chromosomal blaAmpC by quantitative RT-PCR. Alteration of the blaAmpC promoter was studied by PCR and sequencing. We identified 243 (1.1%) AmpC-EC strains out of 21 563 clinical isolates. Three cases with strains carrying ESBLs, 18 strains that were considered due to colonization and 8 cases lost to clinical follow-up were excluded. Finally, 214 cases were included in the analysis. Ninety-one cases (42.5%) and 269 (62.8%) controls were strictly community acquired (P < 0.001). Thirty-five (16.3%) cases and 186 controls (43.5%) did not have any identifiable risk factor (P < 0.001). Among cases, 158 (73.8%) were found to harbour an acquired AmpC (73.4% CMY-2). Previous use of fluoroquinolones [OR 2.6 (95% CI 1.12-3.36); P = 0.008] was independently associated with AmpC-EC in the multivariate analysis. Prevalence of AmpC in E. coli remains low in our area. Plasmid acquisition (CMY type) represents the main mechanism of AmpC production. A high proportion of community-acquired isolates and patients with no identifiable risk factors were found. Previous use of fluoroquinolones was identified as a risk factor. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Quantitative analysis of microbial contamination in private drinking water supply systems.

PubMed

Allevi, Richard P; Krometis, Leigh-Anne H; Hagedorn, Charles; Benham, Brian; Lawrence, Annie H; Ling, Erin J; Ziegler, Peter E

2013-06-01

Over one million households rely on private water supplies (e.g. well, spring, cistern) in the Commonwealth of Virginia, USA. The present study tested 538 private wells and springs in 20 Virginia counties for total coliforms (TCs) and Escherichia coli along with a suite of chemical contaminants. A logistic regression analysis was used to investigate potential correlations between TC contamination and chemical parameters (e.g. NO3(-), turbidity), as well as homeowner-provided survey data describing system characteristics and perceived water quality. Of the 538 samples collected, 41% (n = 221) were positive for TCs and 10% (n = 53) for E. coli. Chemical parameters were not statistically predictive of microbial contamination. Well depth, water treatment, and farm location proximate to the water supply were factors in a regression model that predicted presence/absence of TCs with 74% accuracy. Microbial and chemical source tracking techniques (Bacteroides gene Bac32F and HF183 detection via polymerase chain reaction and optical brightener detection via fluorometry) identified four samples as likely contaminated with human wastewater.

Spatiotemporal Analysis of Microbiological Contamination in New York State Produce Fields following Extensive Flooding from Hurricane Irene, August 2011.

PubMed

Bergholz, Peter W; Strawn, Laura K; Ryan, Gina T; Warchocki, Steven; Wiedmann, Martin

2016-03-01

Although flooding introduces microbiological, chemical, and physical hazards onto croplands, few data are available on the spatial extent, patterns, and development of contamination over time postflooding. To address this paucity of information, we conducted a spatially explicit study of Escherichia coli and Salmonella contamination prevalence and genetic diversity in produce fields after the catastrophic flooding that occurred in New England during 2011. Although no significant differences were detected between the two participating farms, both random forest and logistic regression revealed changes in the spatial pattern of E. coli contamination in drag swab samples over time. Analyses also indicated that E. coli detection was associated with changes in farm management to remediate the land after flooding. In particular, E. coli was widespread in drag swab samples at 21 days postflooding, but the spatial pattern changed by 238 days postflooding such that E. coli was then most prevalent in close proximity to surface water features. The combined results of several population genetics analyses indicated that over time postflooding E. coli populations on the farms (i) changed in composition and (ii) declined overall. Salmonella was primarily detected in surface water features, but some Salmonella strains were isolated from soil and drag swab samples at 21 and 44 days postflooding. Although postflood contamination and land management responses should always be evaluated in the context of each unique farm landscape, our results provide quantitative data on the general patterns of contamination after flooding and support the practice of establishing buffer zones between flood-contaminated cropland and harvestable crops in produce fields.

Methylation-independent adaptation in chemotaxis of Escherichia coli involves acetylation-dependent speed adaptation.

PubMed

Baron, Szilvia; Afanzar, Oshri; Eisenbach, Michael

2017-01-01

Chemoreceptor methylation and demethylation has been shown to be at the core of the adaptation mechanism in Escherichia coli chemotaxis. Nevertheless, mutants lacking the methylation machinery can adapt to some extent. Here we carried out an extensive quantitative analysis of chemotactic and chemokinetic methylation-independent adaptation. We show that partial or complete adaptation of the direction of flagellar rotation and the swimming speed in the absence of the methylation machinery each occurs in a small fraction of cells. Furthermore, deletion of the main enzyme responsible for acetylation of the signaling molecule CheY prevented speed adaptation but not adaptation of the direction of rotation. These results suggest that methylation-independent adaptation in bacterial chemotaxis involves chemokinetic adaptation, which is dependent on CheY acetylation. © 2016 Federation of European Biochemical Societies.

Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study.

PubMed

Liu, Jie; Platts-Mills, James A; Juma, Jane; Kabir, Furqan; Nkeze, Joseph; Okoi, Catherine; Operario, Darwin J; Uddin, Jashim; Ahmed, Shahnawaz; Alonso, Pedro L; Antonio, Martin; Becker, Stephen M; Blackwelder, William C; Breiman, Robert F; Faruque, Abu S G; Fields, Barry; Gratz, Jean; Haque, Rashidul; Hossain, Anowar; Hossain, M Jahangir; Jarju, Sheikh; Qamar, Farah; Iqbal, Najeeha Talat; Kwambana, Brenda; Mandomando, Inacio; McMurry, Timothy L; Ochieng, Caroline; Ochieng, John B; Ochieng, Melvin; Onyango, Clayton; Panchalingam, Sandra; Kalam, Adil; Aziz, Fatima; Qureshi, Shahida; Ramamurthy, Thandavarayan; Roberts, James H; Saha, Debasish; Sow, Samba O; Stroup, Suzanne E; Sur, Dipika; Tamboura, Boubou; Taniuchi, Mami; Tennant, Sharon M; Toema, Deanna; Wu, Yukun; Zaidi, Anita; Nataro, James P; Kotloff, Karen L; Levine, Myron M; Houpt, Eric R

2016-09-24

Diarrhoea is the second leading cause of mortality in children worldwide, but establishing the cause can be complicated by diverse diagnostic approaches and varying test characteristics. We used quantitative molecular diagnostic methods to reassess causes of diarrhoea in the Global Enteric Multicenter Study (GEMS). GEMS was a study of moderate to severe diarrhoea in children younger than 5 years in Africa and Asia. We used quantitative real-time PCR (qPCR) to test for 32 enteropathogens in stool samples from cases and matched asymptomatic controls from GEMS, and compared pathogen-specific attributable incidences with those found with the original GEMS microbiological methods, including culture, EIA, and reverse-transcriptase PCR. We calculated revised pathogen-specific burdens of disease and assessed causes in individual children. We analysed 5304 sample pairs. For most pathogens, incidence was greater with qPCR than with the original methods, particularly for adenovirus 40/41 (around five times), Shigella spp or enteroinvasive Escherichia coli (EIEC) and Campylobactor jejuni o C coli (around two times), and heat-stable enterotoxin-producing E coli ([ST-ETEC] around 1·5 times). The six most attributable pathogens became, in descending order, Shigella spp, rotavirus, adenovirus 40/41, ST-ETEC, Cryptosporidium spp, and Campylobacter spp. Pathogen-attributable diarrhoeal burden was 89·3% (95% CI 83·2-96·0) at the population level, compared with 51·5% (48·0-55·0) in the original GEMS analysis. The top six pathogens accounted for 77·8% (74·6-80·9) of all attributable diarrhoea. With use of model-derived quantitative cutoffs to assess individual diarrhoeal cases, 2254 (42·5%) of 5304 cases had one diarrhoea-associated pathogen detected and 2063 (38·9%) had two or more, with Shigella spp and rotavirus being the pathogens most strongly associated with diarrhoea in children with mixed infections. A quantitative molecular diagnostic approach improved population-level and case-level characterisation of the causes of diarrhoea and indicated a high burden of disease associated with six pathogens, for which targeted treatment should be prioritised. Bill & Melinda Gates Foundation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Low-level lasers and mRNA levels of reference genes used in Escherichia coli

NASA Astrophysics Data System (ADS)

Teixeira, A. F.; Machado, Y. L. R. C.; Fonseca, A. S.; Mencalha, A. L.

2016-11-01

Low-level lasers are widely used for the treatment of diseases and antimicrobial photodynamic therapy. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is widely used to evaluate mRNA levels and output data from a target gene are commonly relative to a reference mRNA that cannot vary according to treatment. In this study, the level of reference genes from Escherichia coli exposed to red or infrared lasers at different fluences was evaluated. E. coli AB1157 cultures were exposed to red (660 nm) and infrared (808 nm) lasers, incubated (20 min, 37 °C), the total RNA was extracted, and cDNA synthesis was performed to evaluate mRNA levels from arcA, gyrA and rpoA genes by RT-qPCR. Melting curves and agarose gel electrophoresis were carried out to evaluate specific amplification. Data were analyzed by geNorm, NormFinder and BestKeeper. The melting curve and agarose gel electrophoresis showed specific amplification. Although mRNA levels from arcA, gyrA or rpoA genes presented no significant variations trough a traditional statistical analysis, Excel-based tools revealed that these reference genes are not suitable for E. coli cultures exposed to lasers. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from arcA, gyrA and rpoA in E. coli cells.

Cadmium resistance mechanism in Escherichia coli P4 and its potential use to bioremediate environmental cadmium.

PubMed

Khan, Zaman; Nisar, Muhammad Atif; Hussain, Syed Zajif; Arshad, Muhammad Nauman; Rehman, Abdul

2015-12-01

A cadmium-resistant bacterium was isolated from industrial wastewater and identified as Escherichia coli (dubbed as P4) on the basis of morphological, biochemical tests and 16S rRNA ribotyping. It showed optimum growth at 30 °C and pH 7. E. coli P4 found to resist Cd(+2) (10.6 mM) as well as Zn(+2) (4.4 mM), Pb(+2) (17 mM), Cu(+2) (3.5 mM), Cr(+6) (4.4 mM), As(+2) (10.6 mM), and Hg(+2) (0.53 mM). It could remove 18.8, 37, and 56 % Cd(+2) from aqueous medium after 48, 96, and 144 h, respectively. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), and Energy-dispersive X-ray (EDX) analysis also confirmed the biosorption of Cd(+2) by E. coli P4. However, temperature and pH were found to be the most critical factors in biosorption of Cd(+2) by E. coli P4. Cd(+2) stress altered E. coli P4 cell physiology analyzed by measuring glutathione (GSH) and non-protein thiol (cysteine) levels which were increased up to 130 and 48 %, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) showed alteration in the expression levels of ftsZ, mutS, clpB, ef-tu, and dnaK genes in the presence of Cd(+2). Total protein profiles of E. coli P4 in the absence and presence of Cd(+2) were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which showed remarkable difference in the banding pattern. czcB gene, a component of czcCBA operon, was amplified from genomic DNA which suggested the chromosomal-borne Cd(+2) resistance in E. coli P4. Furthermore, it harbors smtAB gene which plays a significant role in Cd(+2) resistance.

Non-labeled quantitative proteomic comparison identifies differences in acid resistance between Escherichia coli O157:H7 curli production variants

USDA-ARS?s Scientific Manuscript database

To understand the nature and capability of a bacterial strain to tolerate environmental conditions it is necessary to be able to identify and measure the proteins that they are expressing in any given situation. In this research, the entire protein complements produced by Escherichia coli O157:H7 s...

Quantitative phase imaging of cell division in yeast cells and E.coli using digital holographic microscopy

NASA Astrophysics Data System (ADS)

Pandiyan, Vimal Prabhu; John, Renu

2015-12-01

Digital holographic microscope (DHM) is an emerging quantitative phase imaging technique with unique imaging scales and resolutions leading to multitude of applications. DHM is promising as a novel investigational and applied tool for cell imaging, studying the morphology and real time dynamics of cells and a number of related applications. The use of numerical propagation and computational digital optics offer unique flexibility to tune the depth of focus, and compensate for image aberrations. In this work, we report imaging the dynamics of cell division in E.coli and yeast cells using a DHM platform. We demonstrate 3-D and depth imaging as well as reconstruction of phase profiles of E.coli and yeast cells using the system. We record a digital hologram of E.coli and yeast cells and reconstruct the image using Fresnel propagation algorithm. We also use aberration compensation algorithms for correcting the aberrations that are introduced by the microscope objective in the object path using linear least square fitting techniques. This work demonstrates the strong potential of a DHM platform in 3-D live cell imaging, fast clinical quantifications and pathological applications.

Quantitative Image Restoration in Bright Field Optical Microscopy.

PubMed

Gutiérrez-Medina, Braulio; Sánchez Miranda, Manuel de Jesús

2017-11-07

Bright field (BF) optical microscopy is regarded as a poor method to observe unstained biological samples due to intrinsic low image contrast. We introduce quantitative image restoration in bright field (QRBF), a digital image processing method that restores out-of-focus BF images of unstained cells. Our procedure is based on deconvolution, using a point spread function modeled from theory. By comparing with reference images of bacteria observed in fluorescence, we show that QRBF faithfully recovers shape and enables quantify size of individual cells, even from a single input image. We applied QRBF in a high-throughput image cytometer to assess shape changes in Escherichia coli during hyperosmotic shock, finding size heterogeneity. We demonstrate that QRBF is also applicable to eukaryotic cells (yeast). Altogether, digital restoration emerges as a straightforward alternative to methods designed to generate contrast in BF imaging for quantitative analysis. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Quantitative assessment of the blood-brain barrier opening caused by Streptococcus agalactiae hyaluronidase in a BALB/c mouse model.

PubMed

Luo, Su; Cao, Qing; Ma, Ke; Wang, Zhaofei; Liu, Guangjin; Lu, Chengping; Liu, Yongjie

2017-10-19

Streptococcus agalactiae is a pathogen causing meningitis in animals and humans. However, little is known about the entry of S. agalactiae into brain tissue. In this study, we developed a BALB/c mouse model based on the intravenous injection of β-galactosidase-positive Escherichia coli M5 as an indicator of blood-brain barrier (BBB) opening. Under physiological conditions, the BBB is impermeable to E. coli M5. In pathological conditions caused by S. agalactiae, E. coli M5 is capable of penetrating the brain through a disrupted BBB. The level of BBB opening can be assessed by quantitative measurement of E. coli M5 loads per gram of brain tissue. Further, we used the model to evaluate the role of S. agalactiae hyaluronidase in BBB opening. The inactivation of hylB gene encoding a hyaluronidase, HylB, resulted in significantly decreased E. coli M5 colonization, and the intravenous injection of purified HylB protein induced BBB opening in a dose-dependent manner. This finding verified the direct role of HylB in BBB invasion and traversal, and further demonstrated the practicability of the in vivo mouse model established in this study. This model will help to understand the S. agalactiae-host interactions that are involved in this bacterial traversal of the BBB and to develop efficacious strategies to prevent central nervous system infections.

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.

The evolution of metabolic networks of E. coli

PubMed Central

2011-01-01

Background Despite the availability of numerous complete genome sequences from E. coli strains, published genome-scale metabolic models exist only for two commensal E. coli strains. These models have proven useful for many applications, such as engineering strains for desired product formation, and we sought to explore how constructing and evaluating additional metabolic models for E. coli strains could enhance these efforts. Results We used the genomic information from 16 E. coli strains to generate an E. coli pangenome metabolic network by evaluating their collective 76,990 ORFs. Each of these ORFs was assigned to one of 17,647 ortholog groups including ORFs associated with reactions in the most recent metabolic model for E. coli K-12. For orthologous groups that contain an ORF already represented in the MG1655 model, the gene to protein to reaction associations represented in this model could then be easily propagated to other E. coli strain models. All remaining orthologous groups were evaluated to see if new metabolic reactions could be added to generate a pangenome-scale metabolic model (iEco1712_pan). The pangenome model included reactions from a metabolic model update for E. coli K-12 MG1655 (iEco1339_MG1655) and enabled development of five additional strain-specific genome-scale metabolic models. These additional models include a second K-12 strain (iEco1335_W3110) and four pathogenic strains (two enterohemorrhagic E. coli O157:H7 and two uropathogens). When compared to the E. coli K-12 models, the metabolic models for the enterohemorrhagic (iEco1344_EDL933 and iEco1345_Sakai) and uropathogenic strains (iEco1288_CFT073 and iEco1301_UTI89) contained numerous lineage-specific gene and reaction differences. All six E. coli models were evaluated by comparing model predictions to carbon source utilization measurements under aerobic and anaerobic conditions, and to batch growth profiles in minimal media with 0.2% (w/v) glucose. An ancestral genome-scale metabolic model based on conserved ortholog groups in all 16 E. coli genomes was also constructed, reflecting the conserved ancestral core of E. coli metabolism (iEco1053_core). Comparative analysis of all six strain-specific E. coli models revealed that some of the pathogenic E. coli strains possess reactions in their metabolic networks enabling higher biomass yields on glucose. Finally the lineage-specific metabolic traits were compared to the ancestral core model predictions to derive new insight into the evolution of metabolism within this species. Conclusion Our findings demonstrate that a pangenome-scale metabolic model can be used to rapidly construct additional E. coli strain-specific models, and that quantitative models of different strains of E. coli can accurately predict strain-specific phenotypes. Such pangenome and strain-specific models can be further used to engineer metabolic phenotypes of interest, such as designing new industrial E. coli strains. PMID:22044664

Evaluation of Oxidative Metabolism in Leukocytes during Phagocytosis of Escherichia coli Carrying Genetic Constructs soxS::lux or katG::lux.

PubMed

Karimov, I F; Deryabin, D G; Karimova, D N; Subbotina, T Yu; Manukhov, I V

2016-06-01

We studied ROS generation by human peripheral blood monocytes and granulocytes during phagocytosis of Escherichia coli soxS::lux or katG::lux responding by luminescence (bioluminescence) to the development of oxidative stress. Initially high sensitivity of the bioluminescent reaction of E. coli katG::lux strain to the effects of model ROS (KO2 and H2O2) and pronounced induction of luminescence upon contact with granulocytes, whereas E. coli soxS::lux demonstrated less pronounced reaction to chemical oxidants and bioluminescence was observed primarily upon contact with monocytes. A correlation was found between quantitative characteristics of E. coli katG::lux bioluminescence and luminol-dependent chemiluminescence of leukocytes in some patients, but no dependence of this kind was noted for E. coli soxS::lux. The results can provide experimental substantiation of a new approach for evaluation of ROS production by leukocytes during phagocytosis and choosing the optimal object for these studies.

Effect of alginate microencapsulation on the catalytic efficiency and in vitro enzyme-prodrug therapeutic efficacy of cytosine deaminase and of recombinant E. coli expressing cytosine deaminase.

PubMed

Funaro, Michael G; Nemani, Krishnamurthy V; Chen, Zhihang; Bhujwalla, Zaver M; Griswold, Karl E; Gimi, Barjor

2016-02-01

Cytosine deaminase (CD) catalyses the enzymatic conversion of the non-toxic prodrug 5-fluorocytosine (5-FC) to the potent chemotherapeutic form, 5-fluorouracil (5-FU). Intratumoral delivery of CD localises chemotherapy dose while reducing systemic toxicity. Encapsulation in biocompatible microcapsules immunoisolates CD and protects it from degradation. We report on the effect of alginate encapsulation on the catalytic and functional activity of isolated CD and recombinant E. coli engineered to express CD (E. coli(CD)). Alginate microcapsules containing either CD or Escherichia coli(CD) were prepared using ionotropic gelation. Conversion of 5-FC to 5-FU was quantitated in unencapsulated and encapsulated CD/E. coli(CD) using spectrophotometry, with a slower rate of conversion observed following encapsulation. Both encapsulated CD/5-FC and E. coli(CD)/5-FC resulted in cell kill and reduced proliferation of 9 L rat glioma cells, which was comparable to direct 5-FU treatment. Our results show that encapsulation preserves the therapeutic potential of CD and E. coli(CD) is equally effective for enzyme-prodrug therapy.

3D-QSAR studies on the inhibitory activity of trimethoprim analogues against Escherichia coli dihydrofolate reductase.

PubMed

Vijayaraj, Ramadoss; Devi, Mekapothula Lakshmi Vasavi; Subramanian, Venkatesan; Chattaraj, Pratim Kumar

2012-06-01

Three-dimensional quantitative structure activity relationship (3D-QSAR) study has been carried out on the Escherichia coli DHFR inhibitors 2,4-diamino-5-(substituted-benzyl)pyrimidine derivatives to understand the structural features responsible for the improved potency. To construct highly predictive 3D-QSAR models, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods were used. The predicted models show statistically significant cross-validated and non-cross-validated correlation coefficient of r2 CV and r2 nCV, respectively. The final 3D-QSAR models were validated using structurally diverse test set compounds. Analysis of the contour maps generated from CoMFA and CoMSIA methods reveals that the substitution of electronegative groups at the first and second position along with electropositive group at the third position of R2 substitution significantly increases the potency of the derivatives. The results obtained from the CoMFA and CoMSIA study delineate the substituents on the trimethoprim analogues responsible for the enhanced potency and also provide valuable directions for the design of new trimethoprim analogues with improved affinity. © 2012 John Wiley & Sons A/S.

Growth rate regulation of Escherichia coli acetyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis.

PubMed Central

Li, S J; Cronan, J E

1993-01-01

Acetyl coenzyme A (CoA) carboxylase catalyzes the synthesis of malonyl-CoA, the first intermediate of fatty acid synthesis. The Escherichia coli enzyme is encoded by four subunits located at three different positions on the E. coli chromosome. The accBC genes lie in a small operon at min 72, whereas accA and accD are located at min 4.3 and 50, respectively. We examined the expression of the genes that encode the E. coli acetyl-CoA carboxylase subunits (accA, accBC, and accD) under a variety of growth conditions by quantitative Northern (RNA) blot analysis. We found a direct correlation between the levels of transcription of the acc genes and the rate of cellular growth. Consistent results were also obtained upon nutritional upshift and downshift experiments and upon dilution of stationary-phase cultures into fresh media. We also determined the 5' end of the accA and accD mRNAs by primer extension and did transcriptional fusion analysis of the previously reported accBC promoter. Several interesting features were found in the promoter regions of these genes, including a bent DNA sequence and an open reading frame within the unusually long leader mRNA of the accBC operon, potential stem-loop structures in the accA and accD mRNA leader regions, and a stretch of GC-rich sequences followed by AT-rich sequences common to all three promoters. In addition, both accA and accD are located in complex gene clusters. For example, the accA promoter was localized within the upstream polC gene (which encodes the DNA polymerase III catalytic subunit), suggesting that additional regulatory mechanisms exist. Images PMID:7678242

Transcriptional analysis of the Escherichia coli ColV-Ia plasmid pS88 during growth in human serum and urine.

PubMed

Lemaître, Chloé; Bidet, Philippe; Bingen, Edouard; Bonacorsi, Stéphane

2012-06-21

The sequenced O45:K1:H7 Escherichia coli meningitis strain S88 harbors a large virulence plasmid. To identify possible genetic determinants of pS88 virulence, we examined the transcriptomes of 88 plasmidic ORFs corresponding to known and putative virulence genes, and 35 ORFs of unknown function. Quantification of plasmidic transcripts was obtained by quantitative real-time reverse transcription of extracted RNA, normalized on three housekeeping genes. The transcriptome of E. coli strain S88 grown in human serum and urine ex vivo were compared to that obtained during growth in Luria Bertani broth, with and without iron depletion. We also analyzed the transcriptome of a pS88-like plasmid recovered from a neonate with urinary tract infection. The transcriptome obtained after ex vivo growth in serum and urine was very similar to those obtained in iron-depleted LB broth. Genes encoding iron acquisition systems were strongly upregulated. ShiF and ORF 123, two ORFs encoding protein with hypothetical function and physically linked to aerobactin and salmochelin loci, respectively, were also highly expressed in iron-depleted conditions and may correspond to ancillary iron acquisition genes. Four ORFs were induced ex vivo, independently of the iron concentration. Other putative virulence genes such as iss, etsC, ompTp and hlyF were not upregulated in any of the conditions studied. Transcriptome analysis of the pS88-like plasmid recovered in vivo showed a similar pattern of induction but at much higher levels. We identify new pS88 genes potentially involved in the growth of E. coli meningitis strain S88 in human serum and urine.

QPROT: Statistical method for testing differential expression using protein-level intensity data in label-free quantitative proteomics.

PubMed

Choi, Hyungwon; Kim, Sinae; Fermin, Damian; Tsou, Chih-Chiang; Nesvizhskii, Alexey I

2015-11-03

We introduce QPROT, a statistical framework and computational tool for differential protein expression analysis using protein intensity data. QPROT is an extension of the QSPEC suite, originally developed for spectral count data, adapted for the analysis using continuously measured protein-level intensity data. QPROT offers a new intensity normalization procedure and model-based differential expression analysis, both of which account for missing data. Determination of differential expression of each protein is based on the standardized Z-statistic based on the posterior distribution of the log fold change parameter, guided by the false discovery rate estimated by a well-known Empirical Bayes method. We evaluated the classification performance of QPROT using the quantification calibration data from the clinical proteomic technology assessment for cancer (CPTAC) study and a recently published Escherichia coli benchmark dataset, with evaluation of FDR accuracy in the latter. QPROT is a statistical framework with computational software tool for comparative quantitative proteomics analysis. It features various extensions of QSPEC method originally built for spectral count data analysis, including probabilistic treatment of missing values in protein intensity data. With the increasing popularity of label-free quantitative proteomics data, the proposed method and accompanying software suite will be immediately useful for many proteomics laboratories. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of different growth conditions on the discrimination of three bacteria by pyrolysis gas-liquid chromatography.

PubMed Central

Gutteridge, C S; Norris, J R

1980-01-01

High-resolution pyrolysis gas-liquid chromatography was applied to three bacteria (Escherichia coli NCTC 9001, Pseudomonas putida (NCIB 9494, and Staphylococcus aureus NCTC 8532) grown under a variety of conditions. Changing the culture medium drastically altered the quantitative aspects of the pyrograms of all three organisms, but the effects of culture time and incubation temperature were less severe. Mathematical analysis of the relative peak heights showed that four peaks could be used to discriminate the three bacteria however they were cultured. PMID:6999989

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

PubMed

Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

2015-11-20

The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

Following Drug Uptake and Reactions inside Escherichia coli Cells by Raman Microspectroscopy

PubMed Central

2015-01-01

Raman microspectroscopy combined with Raman difference spectroscopy reveals the details of chemical reactions within bacterial cells. The method provides direct quantitative data on penetration of druglike molecules into Escherichia coli cells in situ along with the details of drug–target reactions. With this label-free technique, clavulanic acid and tazobactam can be observed as they penetrate into E. coli cells and subsequently inhibit β-lactamase enzymes produced within these cells. When E. coli cells contain a β-lactamase that forms a stable complex with an inhibitor, the Raman signature of the known enamine acyl–enzyme complex is detected. From Raman intensities it is facile to measure semiquantitatively the number of clavulanic acid molecules taken up by the lactamase-free cells during growth. PMID:24901294

Successful Validation of Sample Processing and Quantitative Real-Time PCR Capabilities on the International Space Station

NASA Technical Reports Server (NTRS)

Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Schonfeld, Julie; Tran, Luan

2016-01-01

The WetLab-2 system was developed by NASA Ames Research Center to offer new capabilities to researchers. The system can lyse cells and extract RNA (Ribonucleic Acid) on-orbit from different sample types ranging from microbial cultures to animal tissues. The purified RNA can then either be stabilized for return to Earth or can be used to conduct on-orbit quantitative Reverse Transcriptase PCR (Polymerase Chain Reaction) (qRT-PCR) analysis without the need for sample return. The qRT-PCR results can be downlinked to the ground a few hours after the completion of the run. The validation flight of the WetLab-2 system launched on SpaceX-8 on April 8, 2016. On orbit operations started on April 15th with system setup and was followed by three quantitative PCR runs using an E. coli genomic DNA template pre-loaded at three different concentrations. These runs were designed to discern if quantitative PCR functions correctly in microgravity and if the data is comparable to that from the ground control runs. The flight data showed no significant differences compared to the ground data though there was more variability in the values, this was likely due to the numerous small bubbles observed. The capability of the system to process samples and purify RNA was then validated using frozen samples prepared on the ground. The flight data for both E. coli and mouse liver clearly shows that RNA was successfully purified by our system. The E. coli qRT-PCR run showed successful singleplex, duplex and triplex capability. Data showed high variability in the resulting Cts (Cycle Thresholds [for the PCR]) likely due to bubble formation and insufficient mixing during the procedure run. The mouse liver qRT-PCR run had successful singleplex and duplex reactions and the variability was slightly better as the mixing operation was improved. The ability to purify and stabilize RNA and to conduct qRT-PCR on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. The ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. The WetLab-2 Project is supported by the Research Integration Office in the ISS Program.

Natural descriptions of motor behavior: examples from E. coli and C. elegans.

NASA Astrophysics Data System (ADS)

Ryu, William

2007-03-01

E. coli has a natural behavioral variable - the direction of rotation of its flagellar rotorary motor. Monitoring this one-dimensional behavioral response in reaction to chemical perturbation has been instrumental in the understanding of how E. coli performs chemotaxis at the genetic, physiological, and computational level. Here we apply this experimental strategy to the study of bacterial thermotaxis - a sensory mode that is less well understood. We investigate bacterial thermosensation by studying the motor response of single cells subjected to impulses of heat produced by an IR laser. A simple temperature dependent modification to an existing chemotaxis model can explain the observed temperature response. Higher organisms may have a more complicated behavioral response due to the simple fact that their motions have more degrees of freedom. Here we provide a principled analysis of motor behavior of such an organism -- the roundworm C. elegans. Using tracking video-microscopy we capture a worm's image and extract the skeleton of the shape as a head-to-tail ordered collection of tangent angles sampled along the curve. Applying principal components analysis we show that the space of shapes is remarkably low dimensional, with four dimensions accounting for > 95% of the shape variance. We also show that these dimensions align with behaviorally relevant states. As an application of this analysis we study the thermal response of worms stimulated by laser heating. Our quantitative description of C. elegans movement should prove useful in a wide variety of contexts, from the linking of motor output with neural circuitry to the genetic basis of adaptive behavior.

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.

Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches

PubMed Central

Guedich, Sondés; Puffer-Enders, Barbara; Baltzinger, Mireille; Hoffmann, Guillaume; Da Veiga, Cyrielle; Jossinet, Fabrice; Thore, Stéphane; Bec, Guillaume; Ennifar, Eric; Burnouf, Dominique; Dumas, Philippe

2016-01-01

ABSTRACT Riboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations. PMID:26932506

Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches.

PubMed

Guedich, Sondés; Puffer-Enders, Barbara; Baltzinger, Mireille; Hoffmann, Guillaume; Da Veiga, Cyrielle; Jossinet, Fabrice; Thore, Stéphane; Bec, Guillaume; Ennifar, Eric; Burnouf, Dominique; Dumas, Philippe

2016-01-01

Riboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations.

Kinetics of uropathogenic Escherichia coli metapopulation movement during urinary tract infection.

PubMed

Walters, Matthew S; Lane, M Chelsea; Vigil, Patrick D; Smith, Sara N; Walk, Seth T; Mobley, Harry L T

2012-01-01

The urinary tract is one of the most frequent sites of bacterial infection in humans. Uropathogenic Escherichia coli (UPEC) strains are the leading cause of urinary tract infections (UTIs) and are responsible for greater than 80% of uncomplicated cases in adults. Infection of the urinary tract occurs in an ascending manner, with colonization of the bladder leading to possible kidney infection and bacteremia. The goal of this study was to examine the population dynamics of UPEC in vivo using a murine model of ascending UTI. To track individual UPEC lineages within a host, we constructed 10 isogenic clones of UPEC strain CFT073 by inserting unique signature tag sequences between the pstS and glmS genes at the attTn7 chromosomal site. Mice were transurethrally inoculated with a mixture containing equal numbers of unique clones. After 4 and 48 h, the tags present in the bladders, kidneys, and spleens of infected mice were enumerated using tag-specific primers and quantitative real-time PCR. The results indicated that kidney infection and bacteremia associated with UTI are most likely the result of multiple rounds of ascension and dissemination from motile UPEC subpopulations, with a distinct bottleneck existing between the kidney and bloodstream. The abundance of tagged lineages became more variable as infection progressed, especially after bacterial ascension to the upper urinary tract. Analysis of the population kinetics of UPEC during UTI revealed metapopulation dynamics, with lineages that constantly increased and decreased in abundance as they migrated from one organ to another. Urinary tract infections are some of the most common infections affecting humans, and Escherichia coli is the primary cause in most uncomplicated cases. These infections occur in an ascending manner, with bacteria traveling from the bladder to the kidneys and potentially the bloodstream. Little is known about the spatiotemporal population dynamics of uropathogenic E. coli within a host. Here we describe a novel approach for tracking lineages of isogenic tagged E. coli strains within a murine host by the use of quantitative real-time PCR. Understanding the in vivo population dynamics and the factors that shape the bacterial population may prove to be of significant value in the development of novel vaccines and drug therapies.

Monoclonal antibody passive hemagglutination and capture enzyme-linked immunosorbent assays for direct detection and quantitation of F41 and K99 fimbrial antigens in enterotoxigenic Escherichia coli.

PubMed Central

Raybould, T J; Crouch, C F; Acres, S D

1987-01-01

Production of diarrhea in neonatal calves by enterotoxigenic Escherichia coli depends on its ability to attach to the epithelial cells of the intestine via surface adhesins called pili or fimbriae and to secrete enterotoxins. The most important of these fimbriae are designated K99 and F41. We produced and characterized a murine monoclonal antibody specific to F41. This monoclonal antibody and a K99-specific monoclonal antibody were used to develop sensitive and specific passive hemagglutination and capture enzyme-linked immunosorbent assays (ELISAs) for detection and quantitation of F41 and K99 antigens in E. coli cultures and culture supernatants. The capture ELISA systems exhibited excellent sensitivity and specificity, whereas the passive hemagglutination systems appeared to be oversensitive. The ability of the capture ELISAs to detect K99 and F41 fimbrial antigens in fecal specimens from calves was evaluated. Fimbrial antigens were detected in six of six specimens from scouring calves but not in four of four specimens from nonscouring calves. PMID:2880866

Modelling the interactions between Pseudomonas putida and Escherichia coli O157:H7 in fish-burgers: use of the lag-exponential model and of a combined interaction index.

PubMed

Speranza, B; Bevilacqua, A; Mastromatteo, M; Sinigaglia, M; Corbo, M R

2010-08-01

The objective of the current study was to examine the interactions between Pseudomonas putida and Escherichia coli O157:H7 in coculture studies on fish-burgers packed in air and under different modified atmospheres (30 : 40 : 30 O(2) : CO(2) : N(2), 5 : 95 O(2) : CO(2) and 50 : 50 O(2) : CO(2)), throughout the storage at 8 degrees C. The lag-exponential model was applied to describe the microbial growth. To give a quantitative measure of the occurring microbial interactions, two simple parameters were developed: the combined interaction index (CII) and the partial interaction index (PII). Under air, the interaction was significant (P < 0.05) only within the exponential growth phase (CII, 1.72), whereas under the modified atmospheres, the interactions were highly significant (P < 0.001) and occurred both in the exponential and in the stationary phase (CII ranged from 0.33 to 1.18). PII values for E. coli O157:H7 were lower than those calculated for Ps. putida. The interactions occurring into the system affected both E. coli O157:H7 and pseudomonads subpopulations. The packaging atmosphere resulted in a key element. The article provides some useful information on the interactions occurring between E. coli O157:H7 and Ps. putida on fish-burgers. The proposed index describes successfully the competitive growth of both micro-organisms, giving also a quantitative measure of a qualitative phenomenon.

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

An Assessment of Coliform Bacteria in Water Sources Near Appalachian Trail Shelters Within the Great Smoky Mountains National Park.

PubMed

Reed, Brian C; Rasnake, Mark S

2016-03-01

Hikers and campers are exposed to risks while in the wilderness. One of these risks is the possibility of contracting an illness, including infectious diarrhea. This project tested for coliform bacteria in water samples taken near popular Appalachian Trail shelters. Water was collected from access points within the Great Smoky Mountains National Park. Samples were collected in sterile bottles and inoculated on a commercially available coliform detection kit for quantitative determination of total coliform and Escherichia coli counts. Water samples were taken during summer and fall seasons. During summer, 7 of 10 samples were positive for coliform bacteria and 6 of those 7 for E coli. The most probable number (MPN) of colony-forming units (CFU) for coliform bacteria ranged from 0 to 489 CFU/100 mL, with the MPN for E coli varying from 0 to 123 CFU/100 mL. These data differed from the fall collection, revealing 3 of 7 samples positive for coliform bacteria and 1 of those 3 for E coli. The MPN of CFU for coliform bacteria in fall samples varied from 0 to 119 CFU/100 mL and 0 to 5 to CFU/100 mL for E coli. Environmental Protection Agency drinking water standards set the standard of 0 CFU/100 mL to be considered safe. This analysis of water samples along the Appalachian Trail emphasizes that the majority of water access points require treatment during the summer season. Coliform burden was not as high through the fall months. These data suggest one infectious disease risk for wilderness travelers. Copyright © 2016 Wilderness Medical Society. Published by Elsevier Inc. All rights reserved.

Comparing rapid and culture indicator bacteria methods at inland lake beaches

USGS Publications Warehouse

Francy, Donna S.; Bushon, Rebecca N.; Brady, Amie M.G.; Kephart, Christopher M.

2013-01-01

A rapid method, quantitative polymerase chain reaction (qPCR), for quantifying indicator bacteria in recreational waters is desirable for public health protection. We report that replacing current Escherichia coli standards with new US Environmental Protection Agency beach action values (BAVs) for enterococci by culture or qPCR may result in more advisories being posted at inland recreational lakes. In this study, concentrations of E. coli and enterococci by culture methods were compared to concentrations of Enterococcus spp. by qPCR at 3 inland lake beaches in Ohio. The E. coli and enterococci culture results were significantly related at all beaches; however, the relations between culture results and Enterococcus spp. qPCR results were not always significant and differed among beaches. All the qPCR results exceeded the new BAV for Enterococcus spp. by qPCR, whereas only 23.7% of culture results for E. coli and 79% of culture results for enterococci exceeded the current standard for E. coli or BAV for enterococci.

Protective effects of Lactobacillus plantarum against epithelial barrier dysfunction of human colon cell line NCM460

PubMed Central

Liu, Zhi-Hua; Shen, Tong-Yi; Zhang, Peng; Ma, Yan-Lei; Moyer, Mary Pat; Qin, Huan-Long

2010-01-01

AIM: To investigate the effects of Lactobacillus plantarum (L. plantarum) in the intestinal permeability and expression of tight junction (TJ) using the normal human colon cell line NCM460. METHODS: Paracellular permeability of NCM460 monolayers was determined by transepithelial electrical resistance and dextran permeability. Expression of TJ proteins in NCM460 cell monolayers was detected by Western blotting and quantitative real-time polymerase chain reaction. RESULTS: L. plantarum played an important role in increasing transepithelial electrical resistance and decreasing the permeability to macromolecules of NCM460 monolayers against the disruption caused by enteropathogenic Escherichia coli (E. coli) or enteroinvasive E. coli. L. plantarum also prevented the decrease in the expression of TJ proteins and F-actin in NCM460 cells. CONCLUSION: L. plantarum can protect against dysfunction of NCM460 intestinal epithelial barrier caused by enteropathogenic E. coli or enteroinvasive E. coli, and thus can be a potential candidate of therapeutic agents for the treatment of intestinal diseases. PMID:21128328

Evaluation of Multiplex Type-Specific Real-Time PCR Assays Using the LightCycler and Joint Biological Agent Identification and Diagnostic System Platforms for Detection and Quantitation of Adult Human Respiratory Adenoviruses

DTIC Science & Technology

2010-04-01

53592), Escherichia coli, Klebsiella pneu- moniae (ATCC 13883), Pseudomonas aeruginosa (ATCC 97), Mycoplasma pneu- moniae, and Legionella pneumophila... Legionella pneumophila. Additionally, when we tested all samples with the multiplex assays, we did not see any cross- reactivity (data not shown...Chlamydophila pneumoniae Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Mycoplasma pneumoniae Legionella pneumophila VOL. 48, 2010

Decrease of energy spilling in Escherichia coli continuous cultures with rising specific growth rate and carbon wasting

PubMed Central

2011-01-01

Background Growth substrates, aerobic/anaerobic conditions, specific growth rate (μ) etc. strongly influence Escherichia coli cell physiology in terms of cell size, biomass composition, gene and protein expression. To understand the regulation behind these different phenotype properties, it is useful to know carbon flux patterns in the metabolic network which are generally calculated by metabolic flux analysis (MFA). However, rarely is biomass composition determined and carbon balance carefully measured in the same experiments which could possibly lead to distorted MFA results and questionable conclusions. Therefore, we carried out both detailed carbon balance and biomass composition analysis in the same experiments for more accurate quantitative analysis of metabolism and MFA. Results We applied advanced continuous cultivation methods (A-stat and D-stat) to continuously monitor E. coli K-12 MG1655 flux and energy metabolism dynamic responses to change of μ and glucose-acetate co-utilisation. Surprisingly, a 36% reduction of ATP spilling was detected with increasing μ and carbon wasting to non-CO2 by-products under constant biomass yield. The apparent discrepancy between constant biomass yield and decline of ATP spilling could be explained by the rise of carbon wasting from 3 to 11% in the carbon balance which was revealed by the discovered novel excretion profile of E. coli pyrimidine pathway intermediates carbamoyl-phosphate, dihydroorotate and orotate. We found that carbon wasting patterns are dependent not only on μ, but also on glucose-acetate co-utilisation capability. Accumulation of these compounds was coupled to the two-phase acetate accumulation profile. Acetate overflow was observed in parallel with the reduction of TCA cycle and glycolysis fluxes, and induction of pentose phosphate pathway. Conclusions It can be concluded that acetate metabolism is one of the major regulating factors of central carbon metabolism. More importantly, our model calculations with actual biomass composition and detailed carbon balance analysis in steady state conditions with -omics data comparison demonstrate the importance of a comprehensive systems biology approach for more advanced understanding of metabolism and carbon re-routing mechanisms potentially leading to more successful metabolic engineering. PMID:21726468

Microbiological risk from minimally processed packaged salads in the Dutch food chain.

PubMed

Pielaat, Annemarie; van Leusden, Frans M; Wijnands, Lucas M

2014-03-01

The objective of this study was to evaluate the microbial hazard associated with the consumption of mixed salads produced under standard conditions. The presence of Salmonella, Campylobacter spp., and Escherichia coli O157 in the Dutch production chain of mixed salads was determined. Microbial prevalence and concentration data from a microbiological surveillance study were used as inputs for the quantitative microbial risk assessment. Chain logistics, production figures, and consumption patterns were combined with the survey data for the risk assessment chain approach. The results of the sample analysis were used to track events from contamination through human illness. Wide 95% confidence intervals around the mean were found for estimated annual numbers of illnesses resulting from the consumption of mixed salads contaminated with Salmonella Typhimurium DT104 (0 to 10,300 cases), Campylobacter spp. (0 to 92,000 cases), or E. coli (0 to 800 cases). The main sources of uncertainty are the lack of decontamination data (i.e., produce washing during processing) and an appropriate dose-response relationship.

Effects of Limited Aeration and of the ArcAB System on Intermediary Pyruvate Catabolism in Escherichia coli

PubMed Central

Alexeeva, Svetlana; de Kort, Bart; Sawers, Gary; Hellingwerf, Klaas J.; de Mattos, M. Joost Teixeira

2000-01-01

The capacity of Escherichia coli to adapt its catabolism to prevailing redox conditions resides mainly in three catabolic branch points involving (i) pyruvate formate-lyase (PFL) and the pyruvate dehydrogenase complex (PDHc), (ii) the exclusively fermentative enzymes and those of the Krebs cycle, and (iii) the alternative terminal cytochrome bd and cytochrome bo oxidases. A quantitative analysis of the relative catabolic fluxes through these pathways is presented for steady-state glucose-limited chemostat cultures with controlled oxygen availability ranging from full aerobiosis to complete anaerobiosis. Remarkably, PFL contributed significantly to the catabolic flux under microaerobic conditions and was found to be active simultaneously with PDHc and cytochrome bd oxidase-dependent respiration. The synthesis of PFL and cytochrome bd oxidase was found to be maximal in the lower microaerobic range but not in a ΔArcA mutant, and we conclude that the Arc system is more active with respect to regulation of these two positively regulated operons during microaerobiosis than during anaerobiosis. PMID:10940038

Quantitative chemical biosensing by bacterial chemotaxis in microfluidic chips.

PubMed

Roggo, Clémence; Picioreanu, Cristian; Richard, Xavier; Mazza, Christian; van Lintel, Harald; van der Meer, Jan Roelof

2018-01-01

Whole-cell bacterial bioreporters are proposed as alternatives to chemical analysis of, for example, pollutants in environmental compartments. Commonly based on reporter gene induction, bioreporters produce a detectable signal within 30 min to a few hours after exposure to the chemical target, which is impractical for applications aiming at a fast response. In an attempt to attain faster readout but maintain flexibility of chemical targeting, we explored the concept for quantitative chemical sensing by bacterial chemotaxis. Chemotaxis was quantified from enrichment of cells across a 600 µm-wide chemical gradient stabilized by parallel flow in a microfluidic chip, further supported by transport and chemotaxis steady state and kinetic modelling. As proof-of-concept, we quantified Escherichia coli chemotaxis towards serine, aspartate and methylaspartate as a function of attractant concentration and exposure time. E. coli chemotaxis enrichment increased sharply between 0 and 10 µM serine, before saturating at 100 µM. The chemotaxis accumulation rate was maximal at 10 µM serine, leading to observable cell enrichment within 5 min. The potential application for biosensing of environmental toxicants was investigated by quantifying chemotaxis of Cupriavidus pinatubonensis JMP134 towards the herbicide 2,4-dichlorophenoxyacetate. Our results show that bacterial chemotaxis can be quantified on a scale of minutes and may be used for developing faster bioreporter assays. © 2017 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

Characterization and antimicrobial performance of nano silver coatings on leather materials

PubMed Central

Lkhagvajav, N.; Koizhaiganova, M.; Yasa, I.; Çelik, E.; Sari, Ö.

2015-01-01

In this study, the characterization and the antimicrobial properties of nano silver (nAg) coating on leather were investigated. For this purpose, turbidity, viscosity and pH of nAg solutions prepared by the sol-gel method were measured. The formation of films from these solutions was characterized according to temperature by Differential Thermal Analysis-Thermogravimetry (DTA-TG) equipment. The surface morphology of treated leathers was observed using Scanning Electron Microscopy (SEM). The antimicrobial performance of nAg coatings on leather materials to the test microorganisms as Escherichia coli , Staphylococcus aureus , Candida albicans and Aspergillius niger was evaluated by the application of qualitative (Agar overlay method) and quantitative (percentage of microbial reduction) tests. According to qualitative test results it was found that 20 μg/cm 2 and higher concentrations of nAg on the leather samples were effective against all microorganisms tested. Moreover, quantitative test results showed that leather samples treated with 20 μg/cm 2 of nAg demonstrated the highest antibacterial activity against E. coli with 99.25% bacterium removal, whereas a 10 μg/cm 2 concentration of nAg on leather was enough to exhibit the excellent percentage reduction against S. aureus of 99.91%. The results are promising for the use of colloidal nano silver solution on lining leather as antimicrobial coating. PMID:26221087

Identification and quantification of virulence factors of enterotoxigenic Escherichia coli by high-resolution melting curve quantitative PCR.

PubMed

Wang, Weilan; Zijlstra, Ruurd T; Gänzle, Michael G

2017-05-15

Diagnosis of enterotoxigenic E. coli (ETEC) associated diarrhea is complicated by the diversity of E.coli virulence factors. This study developed a multiplex quantitative PCR assay based on high-resolution melting curves analysis (HRM-qPCR) to identify and quantify genes encoding five ETEC fimbriae related to diarrhea in swine, i.e. K99, F41, F18, F6 and K88. Five fimbriae expressed by ETEC were amplified in multiple HRM-qPCR reactions to allow simultaneous identification and quantification of five target genes. The assay was calibrated to allow quantification of the most abundant target gene, and validated by analysis of 30 samples obtained from piglets with diarrhea and healthy controls, and comparison to standard qPCR detection. The five amplicons with melting temperatures (Tm) ranging from 74.7 ± 0.06 to 80.5 ± 0.15 °C were well-separated by HRM-qPCR. The area of amplicons under the melting peak correlated linearly to the proportion of the template in the calibration mixture if the proportion exceeded 4.8% (K88) or <1% (all other amplicons). The suitability of the method was evaluated using 30 samples from weaned pigs aged 6-7 weeks; 14 of these animals suffered from diarrhea in consequence of poor sanitary conditions. Genes encoding fimbriae and enterotoxins were quantified by HRM-qPCR and/or qPCR. The multiplex HRM-qPCR allowed accurate analysis when the total gene copy number of targets was more than 1 × 10 5 / g wet feces and the HRM curves were able to simultaneously distinguish fimbriae genes in the fecal samples. The relative quantification of the most abundant F18 based on melting peak area was highly correlated (P < 0.001; r 2  = 0.956) with that of individual qPCR result but the correlation for less abundant fimbriae was much lower. The multiplex HRM assay identifies ETEC virulence factors specifically and efficiently. It correctly indicated the predominant fimbriae type and additionally provides information of presence/ absence of other fimbriae types and it could find broad applications for pathogen diagnosis.

Further refinement of the Escherichia coli brain abscess model in rat.

PubMed

Nazzaro, J M; Pagel, M A; Neuwelt, E A

1992-09-01

The rat brain abscess model provides a substrate for the modeling of delivery of therapeutic agents to intracerebral mass lesions. We now report refinement of the Escherichia coli brain abscess model in rat. A K1 surface antigen-negative E. coli isolated from human blood culture was stereotaxically inoculated into deep brain sites. Histopathologic analyses and quantitative cultures demonstrated the consistent production of lesions. No animal in this consecutive series developed meningitis, ventriculitis or sepsis. By contrast, prior experience with E. coli abscess production resulted in 25% failure rate of abscess production or death from sepsis. This improvement in the model may be attributable to specific characteristics of the bacteria used, modification of the inoculation method or the intracerebral placement technique. The present work suggests a reliable and consistent brain abscess model, which may be further used to study brain suppuration.

Measuring the Viscosity of the Escherichia coli Plasma Membrane Using Molecular Rotors.

PubMed

Mika, Jacek T; Thompson, Alexander J; Dent, Michael R; Brooks, Nicholas J; Michiels, Jan; Hofkens, Johan; Kuimova, Marina K

2016-10-04

The viscosity is a highly important parameter within the cell membrane, affecting the diffusion of small molecules and, hence, controlling the rates of intracellular reactions. There is significant interest in the direct, quantitative assessment of membrane viscosity. Here we report the use of fluorescence lifetime imaging microscopy of the molecular rotor BODIPY C10 in the membranes of live Escherichia coli bacteria to permit direct quantification of the viscosity. Using this approach, we investigated the viscosity in live E. coli cells, spheroplasts, and liposomes made from E. coli membrane extracts. For live cells and spheroplasts, the viscosity was measured at both room temperature (23°C) and the E. coli growth temperature (37°C), while the membrane extract liposomes were studied over a range of measurement temperatures (5-40°C). At 37°C, we recorded a membrane viscosity in live E. coli cells of 950 cP, which is considerably higher than that previously observed in other live cell membranes (e.g., eukaryotic cells, membranes of Bacillus vegetative cells). Interestingly, this indicates that E. coli cells exhibit a high degree of lipid ordering within their liquid-phase plasma membranes. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Semi-quantitative evaluation of fecal contamination potential by human and ruminant sources using multiple lines of evidence

USGS Publications Warehouse

Stoeckel, D.M.; Stelzer, E.A.; Stogner, R.W.; Mau, D.P.

2011-01-01

Protocols for microbial source tracking of fecal contamination generally are able to identify when a source of contamination is present, but thus far have been unable to evaluate what portion of fecal-indicator bacteria (FIB) came from various sources. A mathematical approach to estimate relative amounts of FIB, such as Escherichia coli, from various sources based on the concentration and distribution of microbial source tracking markers in feces was developed. The approach was tested using dilute fecal suspensions, then applied as part of an analytical suite to a contaminated headwater stream in the Rocky Mountains (Upper Fountain Creek, Colorado). In one single-source fecal suspension, a source that was not present could not be excluded because of incomplete marker specificity; however, human and ruminant sources were detected whenever they were present. In the mixed-feces suspension (pet and human), the minority contributor (human) was detected at a concentration low enough to preclude human contamination as the dominant source of E. coli to the sample. Without the semi-quantitative approach described, simple detects of human-associated marker in stream samples would have provided inaccurate evidence that human contamination was a major source of E. coli to the stream. In samples from Upper Fountain Creek the pattern of E. coli, general and host-associated microbial source tracking markers, nutrients, and wastewater-associated chemical detections-augmented with local observations and land-use patterns-indicated that, contrary to expectations, birds rather than humans or ruminants were the predominant source of fecal contamination to Upper Fountain Creek. This new approach to E. coli allocation, validated by a controlled study and tested by application in a relatively simple setting, represents a widely applicable step forward in the field of microbial source tracking of fecal contamination. ?? 2011 Elsevier Ltd.

On-chip quantitative detection of pathogen genes by autonomous microfluidic PCR platform.

PubMed

Tachibana, Hiroaki; Saito, Masato; Shibuya, Shogo; Tsuji, Koji; Miyagawa, Nobuyuki; Yamanaka, Keiichiro; Tamiya, Eiichi

2015-12-15

Polymerase chain reaction (PCR)-based genetic testing has become a routine part of clinical diagnoses and food testing. In these fields, rapid, easy-to-use, and cost-efficient PCR chips are expected to be appeared for providing such testing on-site. In this study, a new autonomous disposable plastic microfluidic PCR chip was created, and was utilized for quantitative detection of pathogenic microorganisms. To control the capillary flow of the following solution in the PCR microchannel, a driving microchannel was newly designed behind the PCR microchannel. This allowed the effective PCR by simply dropping the PCR solution onto the inlet without any external pumps. In order to achieve disposability, injection-molded cyclo-olefin polymer (COP) of a cost-competitive plastic was used for the PCR chip. We discovered that coating the microchannel walls with non-ionic surfactant produced a suitable hydrophilic surface for driving the capillary flow through the 1250-mm long microchannel. As a result, quantitative real-time PCR with the lowest initial concentration of human, Escherichia coli (E. coli), and pathogenic E. coli O157 genomic DNA of 4, 0.0019, 0.031 pg/μl, respectively, was successfully achieved in less than 18 min. Our results indicate that the platform presented in this study provided a rapid, easy-to-use, and low-cost real-time PCR system that could be potentially used for on-site gene testing. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitative real-time PCR-based assessment of tile drainage management influences on bacterial pathogens in tile drainage and groundwater.

PubMed

Liu, Linda; Cloutier, Michel; Craiovan, Emilia; Edwards, Mark; Frey, Steven K; Gottschall, Natalie; Lapen, David R; Sunohara, Mark; Topp, Edward; Khan, Izhar U H

2018-05-15

This study compared the impact of controlled tile drainage (CD) and freely draining (FD) systems on the prevalence and quantitative real-time PCR-based enumeration of four major pathogens including Arcobacter butzleri, Campylobacter jejuni, Campylobacter coli, and Helicobacter pylori in tile- and groundwater following a fall liquid swine manure (LSM) application on clay loam field plots. Although the prevalence of all target pathogens were detected in CD and FD systems, the loads of A. butzleri, C. jejuni, and C. coli were significantly lower in CD tile-water (p<0.05), in relation to FD tile-water. However, concentrations of A. butzleri were significantly greater in CD than FD tile-water (p<0.05). In shallow groundwater (1.2m depth), concentrations of A. butzleri, C. coli, and H. pylori showed no significant difference between CD and FD plots, while C. jejuni concentrations were significantly higher in FD plots (p<0.05). No impact of CD on the H. pylori was observed since quantitative detection in tile- and groundwater was scarce. Although speculative, H. pylori occurrence may have been related to the application of municipal biosolids four years prior to the LSM experiment. Overall, CD can be used to help minimize off-field export of pathogens into surface waters following manure applications to land, thereby reducing waterborne pathogen exposure risks to humans. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR.

PubMed

Guion, Chase E; Ochoa, Theresa J; Walker, Christopher M; Barletta, Francesca; Cleary, Thomas G

2008-05-01

Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx(1) and stx(2) for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli.

Bacteria, viruses, and parasites in an intermittent stream protected from and exposed to pasturing cattle: prevalence, densities, and quantitative microbial risk assessment.

PubMed

Wilkes, G; Brassard, J; Edge, T A; Gannon, V; Jokinen, C C; Jones, T H; Neumann, N; Pintar, K D M; Ruecker, N; Schmidt, P J; Sunohara, M; Topp, E; Lapen, D R

2013-10-15

Over 3500 individual water samples, for 131 sampling times, targeting waterborne pathogens/fecal indicator bacteria were collected during a 7-year period from 4 sites along an intermittent stream running through a small livestock pasture system with and without cattle access-to-stream restriction measures. The study assessed the impact of cattle pasturing/riparian zone protection on: pathogen (bacterial, viral, parasite) occurrence, concentrations of fecal indicators, and quantitative microbial risk assessments (QMRA) of the risk of Cryptosporidium, Giardia and Escherichia coli O157:H7 infection in humans. Methodologies were developed to compute QMRA mean risks on the basis of water samples exhibiting potentially human infectious Cryptosporidium and E. coli based on genotyping Crytosporidium, and E. coli O157:H7 presence/absence information paired with enumerated E. coli. All Giardia spp. were considered infectious. No significant pasturing treatment effects were observed among pathogens, with the exception of Campylobacter spp. and E. coli O157:H7. Campylobacter spp. prevalence significantly decreased downstream through pasture treatments and E. coli O157:H7 was observed in a few instances in the middle of the unrestricted pasture. Densities of total coliform, fecal coliform, and E. coli reduced significantly downstream in the restricted pasture system, but not in the unrestricted system. Seasonal and flow conditions were associated with greater indicator bacteria densities, especially in the summer. Norovirus GII was detected at rates of 7-22% of samples for all monitoring sites, and rotavirus in 0-7% of samples for all monitoring sites; pasture treatment trends were not evident, however. Seasonal and stream flow variables (and their interactions) were relatively more important than pasture treatments for initially stratifying pathogen occurrence and higher fecal indicator bacteria densities. Significant positive associations among fecal indicator bacteria and Campylobacter spp. detection were observed. For QMRA, adjusting for the proportion of Cryptosporidium spp. detected that are infectious for humans reduces downstream risk estimates by roughly one order of magnitude. Using QMRA in this manner provides a more refined estimate of beneficial management practice effects on pathogen exposure risks to humans. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

An electrochemical immunoassay for Escherichia coli O157:H7 using double functionalized Au@Pt/SiO2 nanocomposites and immune magnetic nanoparticles.

PubMed

Ye, Lingxian; Zhao, Guangying; Dou, Wenchao

2018-05-15

A sensitive Point-of-Care Testing (POCT) with Au-Pt bimetallic nanoparticles (Au@Pt) functionalized silica nanoparticle (SiO 2 NPs) and Fe 3 O 4 magnetic nanoparticles (Fe 3 O 4 NPs) was designed for the quantitative detection of Escherichia coli O157:H7 (E. coli O157:H7). The poly-(4-styrenesulfonic acid-co-maleic acid) (PSSMA) as a negatively charged polyelectrolyte can be easily coated on surface of the amino group modified SiO 2 NPs via electrostatic force. PSSMA is also a good stabilizer for water-soluble bimetallic nanostructures. The PSSMA is first time used as a "bridge" to connect the negative charge Au@Pt NPs to the SiO 2 NPs, forming Au@Pt/SiO 2 NPs. Antibody and invertase conjugated Au@Pt/SiO 2 NPs (denoted as Ab/invertase-Au@Pt/SiO 2 NPs) were used as signal labels. Monoclonal antibody against E. coli O157:H7 (Ab) functionalized magnetic nanoparticles (denoted as Ab-Fe 3 O 4 @SiO 2 NPs) were used to enrich and capture the E. coli O157:H7 in positive sample. The immunosensing platform also composed of a personal glucometer (PGM) using for signal readout. Based on this sandwich-type immunoassay, the invertase in the final formed sandwich immunocomplex catalyzed the hydrolysis of sucrose to produce a large amount of glucose for quantitative readout by the PGM. Under optimal conditions, a linear relationship between the glucose concentration and the logarithm of E. coli O157:H7 concentration was obtained in the concentration range from 3.5 × 10 2 to 3.5 × 10 8 CFU mL -1 with a detection limit of 1.83 × 10 2 CFU mL -1 (3σ). This method was used to detect E. coli O157:H7 in spiked milk samples, indicating its potential practical application. This protocol can be applied in various fields of study. Copyright © 2018 Elsevier B.V. All rights reserved.

Microbiological quality of water from the rivers of Curitiba, Paraná State, Brazil, and the susceptibility to antimicrobial drugs and pathogenicity of Escherichia coli.

PubMed

Giowanella, Melissa; Bozza, Angela; do Rocio Dalzoto, Patricia; Dionísio, Jair Alves; Andraus, Sumaia; Guimarães, Edson Luiz Gomes; Pimentel, Ida Chapaval

2015-11-01

Water safety is determined by several markers, and Escherichia coli is one of the most important indicators of water quality. The objective of this study was to evaluate the microbiological parameters in environmental samples of fresh water from rivers of Curitiba and its metropolitan area in Paraná State, Brazil. In addition, we evaluated the pathogenicity and susceptibility to antimicrobial drugs in E. coli. These evaluations were performed by quantitative and qualitative methods employing selective media for isolating thermotolerant coliforms and biochemical tests for identifying E. coli. Pathogenic strains of E. coli were detected by PCR multiplex using specific primers. From the water samples, 494 thermotolerant coliforms were obtained, of which 96 (19.43%) isolates were characterized as E. coli. Three isolates were identified as enteroaggregative E. coli, one as enterotoxigenic E. coli, one as enteropathogenic E. coli, and two carried the Eae virulence gene. E. coli susceptibility to commonly employed antimicrobial drugs was analyzed by the disc diffusion method. The results showed 49 (51.04%) isolates resistant to all the drugs assayed, 16 (16.67%) with an intermediate resistance to all drugs, and 31 (32.29%) intermediately or fully resistant to one or more drugs tested. The highest rate of resistance was observed for tetracycline 30 μg, streptomycin 10 μg, and ceftazidime 30 μg. Detection of E. coli is associated with water contamination by fecal material from humans and warm-blooded animals. The occurrence of resistant strains can be the result of the indiscriminate use of antimicrobial drugs and poor sanitation in the areas assayed.

Insights into the evolution of pathogenicity of Escherichia coli from genomic analysis of intestinal E. coli of Marmota himalayana in Qinghai–Tibet plateau of China

PubMed Central

Lu, Shan; Jin, Dong; Wu, Shusheng; Yang, Jing; Lan, Ruiting; Bai, Xiangning; Liu, Sha; Meng, Qiong; Yuan, Xuejiao; Zhou, Juan; Pu, Ji; Chen, Qiang; Dai, Hang; Hu, Yuanyuan; Xiong, Yanwen; Ye, Changyun; Xu, Jianguo

2016-01-01

Escherichia coli is both of a widespread harmless gut commensal and a versatile pathogen of humans. Domestic animals are a well-known reservoir for pathogenic E. coli. However, studies of E. coli populations from wild animals that have been separated from human activities had been very limited. Here we obtained 580 isolates from intestinal contents of 116 wild Marmot Marmota himalayana from Qinghai–Tibet plateau, China, with five isolates per animal. We selected 125 (hereinafter referred to as strains) from the 580 isolates for genome sequencing, based on unique pulse field gel electrophoresis patterns and at least one isolate per animal. Whole genome sequence analysis revealed that all 125 strains carried at least one and the majority (79.2%) carried multiple virulence genes based on the analysis of 22 selected virulence genes. In particular, the majority of the strains carried virulence genes from different pathovars as potential 'hybrid pathogens'. The alleles of eight virulence genes from the Marmot E. coli were found to have diverged earlier than all known alleles from human and other animal E. coli. Phylogenetic analysis of the 125 Marmot E. coli genomes and 355 genomes selected from 1622 human and other E. coli strains identified two new phylogroups, G and H, both of which diverged earlier than the other phylogroups. Eight of the 12 well-known pathogenic E. coli lineages were found to share a most recent common ancestor with one or more Marmot E. coli strains. Our results suggested that the intestinal E. coli of the Marmots contained a diverse virulence gene pool and is potentially pathogenic to humans. These findings provided a new understanding of the evolutionary origin of pathogenic E. coli. PMID:27924811

Insights into the evolution of pathogenicity of Escherichia coli from genomic analysis of intestinal E. coli of Marmota himalayana in Qinghai-Tibet plateau of China.

PubMed

Lu, Shan; Jin, Dong; Wu, Shusheng; Yang, Jing; Lan, Ruiting; Bai, Xiangning; Liu, Sha; Meng, Qiong; Yuan, Xuejiao; Zhou, Juan; Pu, Ji; Chen, Qiang; Dai, Hang; Hu, Yuanyuan; Xiong, Yanwen; Ye, Changyun; Xu, Jianguo

2016-12-07

Escherichia coli is both of a widespread harmless gut commensal and a versatile pathogen of humans. Domestic animals are a well-known reservoir for pathogenic E. coli. However, studies of E. coli populations from wild animals that have been separated from human activities had been very limited. Here we obtained 580 isolates from intestinal contents of 116 wild Marmot Marmota himalayana from Qinghai-Tibet plateau, China, with five isolates per animal. We selected 125 (hereinafter referred to as strains) from the 580 isolates for genome sequencing, based on unique pulse field gel electrophoresis patterns and at least one isolate per animal. Whole genome sequence analysis revealed that all 125 strains carried at least one and the majority (79.2%) carried multiple virulence genes based on the analysis of 22 selected virulence genes. In particular, the majority of the strains carried virulence genes from different pathovars as potential 'hybrid pathogens'. The alleles of eight virulence genes from the Marmot E. coli were found to have diverged earlier than all known alleles from human and other animal E. coli. Phylogenetic analysis of the 125 Marmot E. coli genomes and 355 genomes selected from 1622 human and other E. coli strains identified two new phylogroups, G and H, both of which diverged earlier than the other phylogroups. Eight of the 12 well-known pathogenic E. coli lineages were found to share a most recent common ancestor with one or more Marmot E. coli strains. Our results suggested that the intestinal E. coli of the Marmots contained a diverse virulence gene pool and is potentially pathogenic to humans. These findings provided a new understanding of the evolutionary origin of pathogenic E. coli.

40 CFR 141.705 - Approved laboratories.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Cryptosporidium analysis by an equivalent State laboratory certification program. (b) E. coli. Any laboratory... coliform or fecal coliform analysis under § 141.74 is approved for E. coli analysis under this subpart when the laboratory uses the same technique for E. coli that the laboratory uses for § 141.74. (c...

40 CFR 141.705 - Approved laboratories.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Cryptosporidium analysis by an equivalent State laboratory certification program. (b) E. coli. Any laboratory... coliform or fecal coliform analysis under § 141.74 is approved for E. coli analysis under this subpart when the laboratory uses the same technique for E. coli that the laboratory uses for § 141.74. (c...

40 CFR 141.705 - Approved laboratories.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Cryptosporidium analysis by an equivalent State laboratory certification program. (b) E. coli. Any laboratory... coliform or fecal coliform analysis under § 141.74 is approved for E. coli analysis under this subpart when the laboratory uses the same technique for E. coli that the laboratory uses for § 141.74. (c...

40 CFR 141.705 - Approved laboratories.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Cryptosporidium analysis by an equivalent State laboratory certification program. (b) E. coli. Any laboratory... coliform or fecal coliform analysis under § 141.74 is approved for E. coli analysis under this subpart when the laboratory uses the same technique for E. coli that the laboratory uses for § 141.74. (c...

40 CFR 141.705 - Approved laboratories.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Cryptosporidium analysis by an equivalent State laboratory certification program. (b) E. coli. Any laboratory... coliform or fecal coliform analysis under § 141.74 is approved for E. coli analysis under this subpart when the laboratory uses the same technique for E. coli that the laboratory uses for § 141.74. (c...

Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples.

PubMed

Youmans, Bonnie P; Ajami, Nadim J; Jiang, Zhi-Dong; Petrosino, Joseph F; DuPont, Herbert L; Highlander, Sarah K

2014-01-01

Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

IrrE, a Global Regulator of Extreme Radiation Resistance in Deinococcus radiodurans, Enhances Salt Tolerance in Escherichia coli and Brassica napus

PubMed Central

Zhou, Zhengfu; Yan, Yongliang; Zhang, Wei; Lu, Wei; Ping, Shuzhen; Dai, Qilin; Yuan, Menglong; Feng, Bin; Hou, Xiaoguang; Zhang, Ying; Ruiqiang; Liu, Tingting; Feng, Lu; Wang, Lei; Chen, Ming; Lin, Min

2009-01-01

Background Globally, about 20% of cultivated land is now affected by salinity. Salt tolerance is a trait of importance to all crops in saline soils. Previous efforts to improve salt tolerance in crop plants have met with only limited success. Bacteria of the genus Deinococcus are known for their ability to survive highly stressful conditions, and therefore possess a unique pool of genes conferring extreme resistance. In Deinococcus radiodurans, the irrE gene encodes a global regulator responsible for extreme radioresistance. Methodology/Principal Findings Using plate assays, we showed that IrrE protected E. coli cells against salt shock and other abiotic stresses such as oxidative, osmotic and thermal shocks. Comparative proteomic analysis revealed that IrrE functions as a switch to regulate different sets of proteins such as stress responsive proteins, protein kinases, glycerol-degrading enzymes, detoxification proteins, and growth-related proteins in E. coli. We also used quantitative RT-PCR to investigate expression of nine selected stress-responsive genes in transgenic and wild-type Brassica napus plants. Transgenic B. napus plants expressing the IrrE protein can tolerate 350 mM NaCl, a concentration that inhibits the growth of almost all crop plants. Conclusions Expression of IrrE, a global regulator for extreme radiation resistance in D. radiodurans, confers significantly enhanced salt tolerance in both E. coli and B. napus. We thus propose that the irrE gene might be used as a potentially promising transgene to improve abiotic stress tolerances in crop plants. PMID:19204796

Modelling growth of Escherichia coli O157:H7 in fresh-cut lettuce submitted to commercial process conditions: chlorine washing and modified atmosphere packaging.

PubMed

Posada-Izquierdo, Guiomar D; Pérez-Rodríguez, Fernando; López-Gálvez, Francisco; Allende, Ana; Selma, María V; Gil, María I; Zurera, Gonzalo

2013-04-01

Fresh-cut iceberg lettuce inoculated with Escherichia coli O157:H7 was submitted to chlorine washing (150 mg/mL) and modified atmosphere packaging on laboratory scale. Populations of E. coli O157:H7 were assessed in fresh-cut lettuce stored at 4, 8, 13 and 16 °C using 6-8 replicates in each analysis point in order to capture experimental variability. The pathogen was able to grow at temperatures ≥8 °C, although at low temperatures, growth data presented a high variability between replicates. Indeed, at 8 °C after 15 days, some replicates did not show growth while other replicates did present an increase. A growth primary model was fitted to the raw growth data to estimate lag time and maximum growth rate. The prediction and confidence bands for the fitted growth models were estimated based on Monte-Carlo method. The estimated maximum growth rates (log cfu/day) corresponded to 0.14 (95% CI: 0.06-0.31), 0.55 (95% CI: 0.17-1.20) and 1.43 (95% CI: 0.82-2.15) for 8, 13 and 16 °C, respectively. A square-root secondary model was satisfactorily derived from the estimated growth rates (R(2) > 0.80; Bf = 0.97; Af = 1.46). Predictive models and data obtained in this study are intended to improve quantitative risk assessment studies for E. coli O157:H7 in leafy green products. Copyright © 2012. Published by Elsevier Ltd.

Effect of Lactobacillus acidophilus and Bifidobacterium bifidum supplementation to standard triple therapy on Helicobacter pylori eradication and dynamic changes in intestinal flora.

PubMed

Wang, Yu-huan; Huang, Ying

2014-03-01

To investigate Lactobacillus acidophilus (L. acidophilus) and Bifidobacterium bifidum (B. bifidum) supplementation to triple therapy for Helicobacter pylori (H. pylori) eradication and dynamic changes in intestinal flora in children with H. pylori infection. One hundred H. pylori-infected children were randomly assigned to two groups: treatment group (n = 43), standard triple anti-H. pylori therapy plus probiotics of L. acidophilus and B. bifidum for 2 weeks followed by taking probiotics for another 4 weeks; control group (n = 45), standard triple anti-H. pylori therapy for 6 weeks. After 6-week treatment, ¹³C-urease breath test was performed and side effects were monitored during the observation period. Quantitative PCR with 16S rRNA-gene-targeted species-specific primers was carried out for the analysis of human intestinal B. bifidum, L. acidophilus, and Escherichia coli (E. coli). As expected, treatment group could significantly enhance the H. pylori eradication rate (83.7 vs. 64.4 %, P < 0.05). B. bifidum, L. acidophilus, and E. coli showed no statistical difference before or after therapy in the treatment group. The number of B. bifidum and L. acidophilus was significantly decreased after 2-week treatment in the control group, but after 6-week treatment it significantly increased and nearly returned to the level before treatment. The number of E. coli increased significantly after 2-week treatment, while after 6-week treatment, it nearly decreased to the level before treatment. L. acidophilus and B. bifidum supplementation is effective for H. pylori eradication compared with triple therapy alone.

Transcriptional analysis reveals the critical role of RNA polymerase-binding transcription factor, DksA, in regulating multi-drug resistance of Escherichia coli.

PubMed

Wang, Jiawei; Cao, Li; Yang, Xiaowen; Wu, Qingmin; Lu, Lin; Wang, Zhen

2018-05-07

The objective of this study was to comprehensively identify the target genes regulated by the RNA polymerase-binding transcription factor DksA in Escherichia coli, and to clarify the role of DksA in multi-drug resistance. A clinical E. coli strain, E8, was selected to construct the dksA gene deletion mutant by using the Red recombination system. The minimum inhibitory concentrations (MICs) of 12 antibiotics in the E8ΔdksA (mutant) were markedly lower than those in the wild-type strain, E8. Genes differentially expressed in the wild-type and dksA mutant were detected using RNA-Seq and were validated by performing quantitative real-time PCR (qRT-PCR). In total, 168 differentially expressed genes were identified in E8ΔdksA, including 81 up-regulated and 87 down-regulated genes. Many of the genes identified are involved in metabolism, two-component systems, transcriptional regulators, and transport/membrane proteins. Interestingly, genes encoding the transcriptional regulator, MarR, which is known to repress the multiple drug resistance operon, marRAB; MdfA, a transport protein that exhibits multidrug efflux activities; oligopeptide transport system proteins OppA and OppD were among those differentially expressed, and could potentially contribute to the increased drug susceptibility of E8ΔdksA. In conclusion, DksA plays an important role in the multi-drug resistance of this E. coli strain, and directly or indirectly regulates the expression of several genes related to antibiotic resistance. Copyright © 2018. Published by Elsevier B.V.

Automated detection and quantitation of bacterial RNA by using electrical microarrays.

PubMed

Elsholz, B; Wörl, R; Blohm, L; Albers, J; Feucht, H; Grunwald, T; Jürgen, B; Schweder, T; Hintsche, Rainer

2006-07-15

Low-density electrical 16S rRNA specific oligonucleotide microarrays and an automated analysis system have been developed for the identification and quantitation of pathogens. The pathogens are Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis, which are typically involved in urinary tract infections. Interdigitated gold array electrodes (IDA-electrodes), which have structures in the nanometer range, have been used for very sensitive analysis. Thiol-modified oligonucleotides are immobilized on the gold IDA as capture probes. They mediate the specific recognition of the target 16S rRNA by hybridization. Additionally three unlabeled oligonucleotides are hybridized in close proximity to the capturing site. They are supporting molecules, because they improve the RNA hybridization at the capturing site. A biotin labeled detector oligonucleotide is also allowed to hybridize to the captured RNA sequence. The biotin labels enable the binding of avidin alkaline phophatase conjugates. The phosphatase liberates the electrochemical mediator p-aminophenol from its electrically inactive phosphate derivative. The electrical signals were generated by amperometric redox cycling and detected by a unique multipotentiostat. The read out signals of the microarray are position specific current and change over time in proportion to the analyte concentration. If two additional biotins are introduced into the affinity binding complex via the supporting oligonucleotides, the sensitivity of the assays increase more than 60%. The limit of detection of Escherichia coli total RNA has been determined to be 0.5 ng/microL. The control of fluidics for variable assay formats as well as the multichannel electrical read out and data handling have all been fully automated. The fast and easy procedure does not require any amplification of the targeted nucleic acids by PCR.

A Novel Quantitative Hemolytic Assay Coupled with Restriction Fragment Length Polymorphisms Analysis Enabled Early Diagnosis of Atypical Hemolytic Uremic Syndrome and Identified Unique Predisposing Mutations in Japan

PubMed Central

Yoshida, Yoko; Miyata, Toshiyuki; Matsumoto, Masanori; Shirotani-Ikejima, Hiroko; Uchida, Yumiko; Ohyama, Yoshifumi; Kokubo, Tetsuro; Fujimura, Yoshihiro

2015-01-01

For thrombotic microangiopathies (TMAs), the diagnosis of atypical hemolytic uremic syndrome (aHUS) is made by ruling out Shiga toxin-producing Escherichia coli (STEC)-associated HUS and ADAMTS13 activity-deficient thrombotic thrombocytopenic purpura (TTP), often using the exclusion criteria for secondary TMAs. Nowadays, assays for ADAMTS13 activity and evaluation for STEC infection can be performed within a few hours. However, a confident diagnosis of aHUS often requires comprehensive gene analysis of the alternative complement activation pathway, which usually takes at least several weeks. However, predisposing genetic abnormalities are only identified in approximately 70% of aHUS. To facilitate the diagnosis of complement-mediated aHUS, we describe a quantitative hemolytic assay using sheep red blood cells (RBCs) and human citrated plasma, spiked with or without a novel inhibitory anti-complement factor H (CFH) monoclonal antibody. Among 45 aHUS patients in Japan, 24% (11/45) had moderate-to-severe (≥50%) hemolysis, whereas the remaining 76% (34/45) patients had mild or no hemolysis (<50%). The former group is largely attributed to CFH-related abnormalities, and the latter group has C3-p.I1157T mutations (16/34), which were identified by restriction fragment length polymorphism (RFLP) analysis. Thus, a quantitative hemolytic assay coupled with RFLP analysis enabled the early diagnosis of complement-mediated aHUS in 60% (27/45) of patients in Japan within a week of presentation. We hypothesize that this novel quantitative hemolytic assay would be more useful in a Caucasian population, who may have a higher proportion of CFH mutations than Japanese patients. PMID:25951460

Paediatric HUS Cases Related to the Consumption of Raw Milk Sold by Vending Machines in Italy: Quantitative Risk Assessment Based on Escherichia coli O157 Official Controls over 7 years.

PubMed

Giacometti, F; Bonilauri, P; Piva, S; Scavia, G; Amatiste, S; Bianchi, D M; Losio, M N; Bilei, S; Cascone, G; Comin, D; Daminelli, P; Decastelli, L; Merialdi, G; Mioni, R; Peli, A; Petruzzelli, A; Tonucci, F; Liuzzo, G; Serraino, A

2017-11-01

A quantitative risk assessment (RA) was developed to estimate haemolytic-uremic syndrome (HUS) cases in paediatric population associated with the consumption of raw milk sold in vending machines in Italy. The historical national evolution of raw milk consumption phenomenon since 2008, when consumer interest started to grow, and after 7 years of marketing adjustment, is outlined. Exposure assessment was based on the official Shiga toxin-producing Escherichia coli O157:H7 (STEC) microbiological records of raw milk samples from vending machines monitored by the regional Veterinary Authorities from 2008 to 2014, microbial growth during storage, consumption frequency of raw milk, serving size, consumption preference and age of consumers. The differential risk considered milk handled under regulation conditions (4°C throughout all phases) and the worst time-temperature field handling conditions detected. In case of boiling milk before consumption, we assumed that the risk of HUS is fixed at zero. The model estimates clearly show that the public health significance of HUS cases due to raw milk STEC contamination depends on the current variability surrounding the risk profile of the food and the consumer behaviour has more impact than milk storage scenario. The estimated HUS cases predicted by our model are roughly in line with the effective STEC O157-associated HUS cases notified in Italy only when the proportion of consumers not boiling milk before consumption is assumed to be 1%. Raw milk consumption remains a source of E. coli O157:H7 for humans, but its overall relevance is likely to have subsided and significant caution should be exerted for temporal, geographical and consumers behaviour analysis. Health education programmes and regulatory actions are required to educate people, primarily children, on other STEC sources. © 2016 Blackwell Verlag GmbH.

Fluorescence resonance energy transfer sensors for quantitative monitoring of pentose and disaccharide accumulation in bacteria

PubMed Central

Kaper, Thijs; Lager, Ida; Looger, Loren L; Chermak, Diane; Frommer, Wolf B

2008-01-01

Background Engineering microorganisms to improve metabolite flux requires detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. These sensors have been applied successfully in mammalian and plant cells but potentially could also be used to monitor steady-state levels of metabolites in microorganisms using fluorimetric assays. Sensors for hexose and pentose carbohydrates could help in the development of fermentative microorganisms, for example, for biofuels applications. Arabinose is one of the carbohydrates to be monitored during biofuels production from lignocellulose, while maltose is an important degradation product of starch that is relevant for starch-derived biofuels production. Results An Escherichia coli expression vector compatible with phage λ recombination technology was constructed to facilitate sensor construction and was used to generate a novel fluorescence resonance energy transfer sensor for arabinose. In parallel, a strategy for improving the sensor signal was applied to construct an improved maltose sensor. Both sensors were expressed in the cytosol of E. coli and sugar accumulation was monitored using a simple fluorimetric assay of E. coli cultures in microtiter plates. In the case of both nanosensors, the addition of the respective ligand led to concentration-dependent fluorescence resonance energy transfer responses allowing quantitative analysis of the intracellular sugar levels at given extracellular supply levels as well as accumulation rates. Conclusion The nanosensor destination vector combined with the optimization strategy for sensor responses should help to accelerate the development of metabolite sensors. The new carbohydrate fluorescence resonance energy transfer sensors can be used for in vivo monitoring of sugar levels in prokaryotes, demonstrating the potential of such sensors as reporter tools in the development of metabolically engineered microbial strains or for real-time monitoring of intracellular metabolite during fermentation. PMID:18522753

Escherichia coli transfer from simulated wildlife feces to lettuce during foliar irrigation: A field study in the Northeastern United States.

PubMed

Weller, Daniel L; Kovac, Jasna; Kent, David J; Roof, Sherry; Tokman, Jeffrey I; Mudrak, Erika; Kowalcyk, Barbara; Oryang, David; Aceituno, Anna; Wiedmann, Martin

2017-12-01

Wildlife intrusion has been associated with pathogen contamination of produce. However, few studies have examined pathogen transfer from wildlife feces to pre-harvest produce. This study was performed to calculate transfer coefficients for Escherichia coli from simulated wildlife feces to field-grown lettuce during irrigation. Rabbit feces inoculated with a 3-strain cocktail of non-pathogenic E. coli were placed in a lettuce field 2.5-72 h before irrigation. Following irrigation, the E. coli concentration on the lettuce was determined. After exclusion of an outlier with high E. coli levels (Most Probable Number = 5.94*10 8 ), the average percent of E. coli in the feces that transferred to intact lettuce heads was 0.0267% (Standard Error [SE] = 0.0172). Log-linear regression showed that significantly more E. coli transferred to outer leaves compared to inner leaves (Effect = 1.3; 95% Confidence Interval = 0.4, 2.1). Additionally, the percent of E. coli that transferred from the feces to the lettuce decreased significantly with time after fecal placement, and as the distance between the lettuce and the feces, and the lettuce and the sprinklers increased. These findings provide key data that may be used in future quantitative risk assessments to identify potential intervention strategies for reducing food safety risks associated with fresh produce. Copyright © 2017 Elsevier Ltd. All rights reserved.

Survival of Escherichia coli on Lettuce under Field Conditions Encountered in the Northeastern United States.

PubMed

Weller, Daniel L; Kovac, Jasna; Roof, Sherry; Kent, David J; Tokman, Jeffrey I; Kowalcyk, Barbara; Oryang, David; Ivanek, Renata; Aceituno, Anna; Sroka, Christopher; Wiedmann, Martin

2017-07-01

Although wildlife intrusion and untreated manure have been associated with microbial contamination of produce, relatively few studies have examined the survival of Escherichia coli on produce under field conditions following contamination (e.g., via splash from wildlife feces). This experimental study was performed to estimate the die-off rate of E. coli on preharvest lettuce following contamination with a fecal slurry. During August 2015, field-grown lettuce was inoculated via pipette with a fecal slurry that was spiked with a three-strain cocktail of rifampin-resistant nonpathogenic E. coli. Ten lettuce heads were harvested at each of 13 time points following inoculation (0, 2.5, 5, and 24 h after inoculation and every 24 h thereafter until day 10). The most probable number (MPN) of E. coli on each lettuce head was determined, and die-off rates were estimated. The relationship between sample time and the log MPN of E. coli per head was modeled using a segmented linear model. This model had a breakpoint at 106 h (95% confidence interval = 69, 142 h) after inoculation, with a daily decrease of 0.70 and 0.19 log MPN for 0 to 106 h and 106 to 240 h following inoculation, respectively. These findings are consistent with die-off rates obtained in similar studies that assessed E. coli survival on produce following irrigation. Overall, these findings provide die-off rates for E. coli on lettuce that can be used in future quantitative risk assessments.

High-sensitivity stable-isotope probing by a quantitative terminal restriction fragment length polymorphism protocol.

PubMed

Andeer, Peter; Strand, Stuart E; Stahl, David A

2012-01-01

Stable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)-TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli, Rhodococcus, Variovorax, and Microbacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.

Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis

PubMed Central

Banowary, Banya; Dang, Van Tuan; Sarker, Subir; Connolly, Joanne H.; Chenu, Jeremy; Groves, Peter; Ayton, Michelle; Raidal, Shane; Devi, Aruna; Vanniasinkam, Thiru; Ghorashi, Seyed A.

2015-01-01

Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates. PMID:26394042

Comparison of Six Chromogenic Agar Media for the Isolation of a Broad Variety of Non-O157 Shigatoxin-Producing Escherichia coli (STEC) Serogroups

PubMed Central

Verhaegen, Bavo; De Reu, Koen; Heyndrickx, Marc; De Zutter, Lieven

2015-01-01

The isolation of non-O157 STEC from food samples has proved to be challenging. The selection of a suitable selective isolation agar remains problematic. The purpose of this study was to qualitatively and quantitatively evaluate six chromogenic agar media for the isolation of STEC: Tryptone Bile X-glucuronide agar (TBX), Rainbow® Agar O157 (RB), Rapid E. coli O157:H7 (RE), Modified MacConkey Agar (mMac), CHROMagarTM STEC (Chr ST) and chromIDTM EHEC (Chr ID). During this study, 45 E. coli strains were used, including 39 STEC strains belonging to 16 different O serogroups and 6 non-STEC E. coli. All E. coli strains were able to grow on TBX and RB, whereas one STEC strain was unable to grow on Chr ID and a number of other STEC strains did not grow on mMac, CHROMagar STEC and Rapid E. coli O157:H7. However, only the latter three agars were selective enough to completely inhibit the growth of the non-STEC E. coli. Our conclusion was that paired use of a more selective agar such as CHROMagar STEC together with a less selective agar like TBX or Chr ID might be the best solution for isolating non-O157 STEC from food. PMID:26090610

Comparison of Six Chromogenic Agar Media for the Isolation of a Broad Variety of Non-O157 Shigatoxin-Producing Escherichia coli (STEC) Serogroups.

PubMed

Verhaegen, Bavo; De Reu, Koen; Heyndrickx, Marc; De Zutter, Lieven

2015-06-17

The isolation of non-O157 STEC from food samples has proved to be challenging. The selection of a suitable selective isolation agar remains problematic. The purpose of this study was to qualitatively and quantitatively evaluate six chromogenic agar media for the isolation of STEC: Tryptone Bile X-glucuronide agar (TBX), Rainbow® Agar O157 (RB), Rapid E. coli O157:H7 (RE), Modified MacConkey Agar (mMac), CHROMagarTM STEC (Chr ST) and chromIDTM EHEC (Chr ID). During this study, 45 E. coli strains were used, including 39 STEC strains belonging to 16 different O serogroups and 6 non-STEC E. coli. All E. coli strains were able to grow on TBX and RB, whereas one STEC strain was unable to grow on Chr ID and a number of other STEC strains did not grow on mMac, CHROMagar STEC and Rapid E. coli O157:H7. However, only the latter three agars were selective enough to completely inhibit the growth of the non-STEC E. coli. Our conclusion was that paired use of a more selective agar such as CHROMagar STEC together with a less selective agar like TBX or Chr ID might be the best solution for isolating non-O157 STEC from food.

Gram Stains: A Resource for Retrospective Analysis of Bacterial Pathogens in Clinical Studies

PubMed Central

Srinivasan, Usha; Ponnaluri, Sreelatha; Villareal, Lisa; Gillespie, Brenda; Wen, Ai; Miles, Arianna; Bucholz, Brigette; Marrs, Carl F.; Iyer, Ram K.; Misra, Dawn; Foxman, Betsy

2012-01-01

We demonstrate the feasibility of using qPCR on DNA extracted from vaginal Gram stain slides to estimate the presence and relative abundance of specific bacterial pathogens. We first tested Gram stained slides spiked with a mix of 108 cfu/ml of Escherichia coli and 105 cfu/ml of Lactobacillus acidophilus. Primers were designed for amplification of total and species-specific bacterial DNA based on 16S ribosomal gene regions. Sample DNA was pre-amplified with nearly full length 16S rDNA ribosomal gene fragment, followed by quantitative PCR with genera and species-specific 16S rDNA primers. Pre-amplification PCR increased the bacterial amounts; relative proportions of Escherichia coli and Lactobacillus recovered from spiked slides remained unchanged. We applied this method to forty two archived Gram stained slides available from a clinical trial of cerclage in pregnant women at high risk of preterm birth. We found a high correlation between Nugent scores based on bacterial morphology of Lactobacillus, Gardenerella and Mobiluncus and amounts of quantitative PCR estimated genus specific DNA (rrn copies) from Gram stained slides. Testing of a convenience sample of eight paired vaginal swabs and Gram stains freshly collected from healthy women found similar qPCR generated estimates of Lactobacillus proportions from Gram stained slides and vaginal swabs. Archived Gram stained slides collected from large scale epidemiologic and clinical studies represent a valuable, untapped resource for research on the composition of bacterial communities that colonize human mucosal surfaces. PMID:23071487

Gram stains: a resource for retrospective analysis of bacterial pathogens in clinical studies.

PubMed

Srinivasan, Usha; Ponnaluri, Sreelatha; Villareal, Lisa; Gillespie, Brenda; Wen, Ai; Miles, Arianna; Bucholz, Brigette; Marrs, Carl F; Iyer, Ram K; Misra, Dawn; Foxman, Betsy

2012-01-01

We demonstrate the feasibility of using qPCR on DNA extracted from vaginal Gram stain slides to estimate the presence and relative abundance of specific bacterial pathogens. We first tested Gram stained slides spiked with a mix of 10(8) cfu/ml of Escherichia coli and 10(5) cfu/ml of Lactobacillus acidophilus. Primers were designed for amplification of total and species-specific bacterial DNA based on 16S ribosomal gene regions. Sample DNA was pre-amplified with nearly full length 16S rDNA ribosomal gene fragment, followed by quantitative PCR with genera and species-specific 16S rDNA primers. Pre-amplification PCR increased the bacterial amounts; relative proportions of Escherichia coli and Lactobacillus recovered from spiked slides remained unchanged. We applied this method to forty two archived Gram stained slides available from a clinical trial of cerclage in pregnant women at high risk of preterm birth. We found a high correlation between Nugent scores based on bacterial morphology of Lactobacillus, Gardenerella and Mobiluncus and amounts of quantitative PCR estimated genus specific DNA (rrn copies) from Gram stained slides. Testing of a convenience sample of eight paired vaginal swabs and Gram stains freshly collected from healthy women found similar qPCR generated estimates of Lactobacillus proportions from Gram stained slides and vaginal swabs. Archived Gram stained slides collected from large scale epidemiologic and clinical studies represent a valuable, untapped resource for research on the composition of bacterial communities that colonize human mucosal surfaces.

Proteomic analysis of enterotoxigenic Escherichia coli (ETEC) in neutral and alkaline conditions.

PubMed

Gonzales-Siles, Lucia; Karlsson, Roger; Kenny, Diarmuid; Karlsson, Anders; Sjöling, Åsa

2017-01-07

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and travelers to endemic areas. Secretion of the heat labile AB 5 toxin (LT) is induced by alkaline conditions. In this study, we determined the surface proteome of ETEC exposed to alkaline conditions (pH 9) as compared to neutral conditions (pH 7) using a LPI Hexalane FlowCell combined with quantitative proteomics. Relative quantitation with isobaric labeling (TMT) was used to compare peptide abundance and their corresponding proteins in multiple samples at MS/MS level. For protein identification and quantification samples were analyzed using either a 1D-LCMS or a 2D-LCMS approach. Strong up-regulation of the ATP synthase operon encoding F1Fo ATP synthase and down-regulation of proton pumping proteins NuoF, NuoG, Ndh and WrbA were detected among proteins involved in regulating the proton and electron transport under alkaline conditions. Reduced expression of proteins involved in osmotic stress was found at alkaline conditions while the Sec-dependent transport over the inner membrane and outer membrane protein proteins such as OmpA and the β-Barrel Assembly Machinery (BAM) complex were up-regulated. ETEC exposed to alkaline environments express a specific proteome profile characterized by up-regulation of membrane proteins and secretion of LT toxin. Alkaline microenvironments have been reported close to the intestinal epithelium and the alkaline proteome may hence represent a better view of ETEC during infection.

Motile bacteria in a critical fluid mixture

NASA Astrophysics Data System (ADS)

Koumakis, Nick; Devailly, Clémence; Poon, Wilson C. K.

2018-06-01

We studied the swimming of Escherichia coli bacteria in the vicinity of the critical point in a solution of the nonionic surfactant C12E5 in buffer solution. In phase-contrast microscopy, each swimming cell produces a transient trail behind itself lasting several seconds. Comparing quantitative image analysis with simulations show that these trails are due to local phase reorganization triggered by differential adsorption. This contrasts with similar trails seen in bacteria swimming in liquid crystals, which are due to shear effects. We show how our trails are controlled, and use them to probe the structure and dynamics of critical fluctuations in the fluid medium.

A whole-cell bioreporter assay for quantitative genotoxicity evaluation of environmental samples.

PubMed

Jiang, Bo; Li, Guanghe; Xing, Yi; Zhang, Dayi; Jia, Jianli; Cui, Zhisong; Luan, Xiao; Tang, Hui

2017-10-01

Whole-cell bioreporters have emerged as promising tools for genotoxicity evaluation, due to their rapidity, cost-effectiveness, sensitivity and selectivity. In this study, a method for detecting genotoxicity in environmental samples was developed using the bioluminescent whole-cell bioreporter Escherichia coli recA::luxCDABE. To further test its performance in a real world scenario, the E. coli bioreporter was applied in two cases: i) soil samples collected from chromium(VI) contaminated sites; ii) crude oil contaminated seawater collected after the Jiaozhou Bay oil spill which occurred in 2013. The chromium(VI) contaminated soils were pretreated by water extraction, and directly exposed to the bioreporter in two phases: aqueous soil extraction (water phase) and soil supernatant (solid phase). The results indicated that both extractable and soil particle fixed chromium(VI) were bioavailable to the bioreporter, and the solid-phase contact bioreporter assay provided a more precise evaluation of soil genotoxicity. For crude oil contaminated seawater, the response of the bioreporter clearly illustrated the spatial and time change in genotoxicity surrounding the spill site, suggesting that the crude oil degradation process decreased the genotoxic risk to ecosystem. In addition, the performance of the bioreporter was simulated by a modified cross-regulation gene expression model, which quantitatively described the DNA damage response of the E. coli bioreporter. Accordingly, the bioluminescent response of the bioreporter was calculated as the mitomycin C equivalent, enabling quantitative comparison of genotoxicities between different environmental samples. This bioreporter assay provides a rapid and sensitive screening tool for direct genotoxicity assessment of environmental samples. Copyright © 2017. Published by Elsevier Ltd.

Estimation of Metabolism Characteristics for Heat-Injured Bacteria Using Dielectrophoretic Impedance Measurement Method

NASA Astrophysics Data System (ADS)

Amako, Eri; Enjoji, Takaharu; Uchida, Satoshi; Tochikubo, Fumiyoshi

Constant monitoring and immediate control of fermentation processes have been required for advanced quality preservation in food industry. In the present work, simple estimation of metabolic states for heat-injured Escherichia coli (E. coli) in a micro-cell was investigated using dielectrophoretic impedance measurement (DEPIM) method. Temporal change in the conductance between micro-gap (ΔG) was measured for various heat treatment temperatures. In addition, the dependence of enzyme activity, growth capacity and membrane situation for E. coli on heat treatment temperature was also analyzed with conventional biological methods. Consequently, a correlation between ΔG and those biological properties was obtained quantitatively. This result suggests that DEPIM method will be available for an effective monitoring technique for complex change in various biological states of microorganisms.

Synergistic effects of Candida and Escherichia coli on gut barrier function.

PubMed

Diebel, L N; Liberati, D M; Diglio, C A; Dulchavsky, S A; Brown, W J

1999-12-01

Disruption of the indigenous gut microflora with overgrowth of gram-negative bacteria and Candida species is common in the critically ill patient. These organisms readily translocate in vitro, which may cause septic complications and organ failure. A synergistic effect between Escherichia coli and C. albicans in polymicrobial infections has been demonstrated. An interaction between these organisms at the mucosal barrier is unknown. Ca(CO2) monolayers were grown to confluence in a two compartment culture system. E. coli and C. albicans or E. coli alone were added to the apical chambers. Secretory immunoglobulin A was added to half of the apical chambers as well. Cell cultures were incubated for a total of 240 minutes. Basal media were sampled at timed intervals for quantitative culture. Monolayer integrity was confirmed by serial measurement of transepithelial electrical resistance. Secretory immunoglobulin A decreased bacterial translocation across Ca(CO2) monolayers challenged with E. coli alone. Transepithelial passage of E. coli was significantly increased by coculture of bacteria with C. albicans. Augmentation of bacterial translocation by Candida occurred even in the presence of secretory immunoglobulin A. Candida colonization of the GI tract may impair mucosal barrier defense against gram-negative bacteria. The clinical role of gut antifungal prophylaxis in protecting against gut derived gram-negative sepsis is speculative.

Women with symptoms of a urinary tract infection but a negative urine culture: PCR-based quantification of Escherichia coli suggests infection in most cases.

PubMed

Heytens, S; De Sutter, A; Coorevits, L; Cools, P; Boelens, J; Van Simaey, L; Christiaens, T; Vaneechoutte, M; Claeys, G

2017-09-01

Our objective was to examine whether or not women with symptoms of a urinary tract infection but with a negative culture (20%-30%) do have an infection. We performed quantitative PCR (qPCR) for Escherichia coli and Staphylococcus saprophyticus, on top of a standard culture, in urine samples from 220 women with dysuria and/or frequency and/or urgency and from 86 women without symptoms. For symptomatic women, qPCR was also carried out for four sexually transmitted agents. In the symptomatic group, 80.9% (178/220) of the urine cultures were positive for any uropathogen and 95.9% (211/220) were E. coli qPCR-positive. For the control group, cultures for E. coli and E. coli qPCR were positive in, respectively, 10.5% (9/86) and 11.6% (10/86). In the symptomatic group, qPCR yielded 19 positive samples for S. saprophyticus qPCR, one positive sample for Mycoplasma genitalium and one for Trichomonas vaginalis. These findings suggest that almost all women with typical urinary complaints and a negative culture still have an infection with E. coli. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Decontamination of a drinking water pipeline system contaminated with adenovirus and Escherichia coli utilizing peracetic acid and chlorine.

PubMed

Kauppinen, Ari; Ikonen, Jenni; Pursiainen, Anna; Pitkänen, Tarja; Miettinen, Ilkka T

2012-09-01

A contaminated drinking water distribution network can be responsible for major outbreaks of infections. In this study, two chemical decontaminants, peracetic acid (PAA) and chlorine, were used to test how a laboratory-scale pipeline system can be cleaned after simultaneous contamination with human adenovirus 40 (AdV40) and Escherichia coli. In addition, the effect of the decontaminants on biofilms was followed as heterotrophic plate counts (HPC) and total cell counts (TCC). Real-time quantitative polymerase chain reaction (qPCR) was used to determine AdV40 and plate counting was used to enumerate E. coli. PAA and chlorine proved to be effective decontaminants since they decreased the levels of AdV40 and E. coli to below method detection limits in both water and biofilms. However, without decontamination, AdV40 remained present in the pipelines for up to 4 days. In contrast, the concentration of cultivable E. coli decreased rapidly in the control pipelines, implying that E. coli may be an inadequate indicator for the presence of viral pathogens. Biofilms responded to the decontaminants by decreased HPCs while TCC remained stable. This indicates that the mechanism of pipeline decontamination by chlorine and PAA is inactivation rather than physical removal of microbes.

Sensitivity to disinfection of bacterial indicator organisms for monitoring the Salmonella Enteritidis status of layer farms after cleaning and disinfection.

PubMed

Dewaele, I; Ducatelle, R; Herman, L; Heyndrickx, M; De Reu, K

2011-06-01

The present study evaluated Escherichia coli, Enterococcus faecalis, and Enterococcus hirae as potential indicator organisms for the possible Salmonella Enteritidis (SE) presence in layer farms after cleaning and disinfection by comparing their susceptibility to disinfection. A quantitative suspension disinfection test according to European Standard EN1656 was performed using disinfection products CID20 and Virocid (both from CID Lines, Ieper, Belgium). In a preliminary test, the sensitivity to both disinfection products was compared between ATCC strains of SE, E. coli, En. faecalis, and En. hirae. The sensitivity of SE to disinfection was most comparable to that of E. coli. A second disinfection test compared the elimination of E. coli to SE ATCC strains as well as field strains. Results showed no significant effect regarding the strain (P > 0.05 for CID20 and Virocid), meaning that no difference was detected in sensitivity toward disinfection. When comparing the sensitivity in general at species level for all concentrations of disinfectant used, no significant difference was found between E. coli and SE in sensitivity to Virocid (P > 0.05). In conclusion, because of its similar response to disinfection in a suspension disinfection test, E. coli could be used as an indicator for possible Salmonella presence after cleaning and disinfection.

Microbial risk in wastewater irrigated lettuce: comparing Escherichia coli contamination from an experimental site with a laboratory approach.

PubMed

Makkaew, P; Miller, M; Fallowfield, H J; Cromar, N J

This study assessed the contamination of Escherichia coli, in lettuce grown with treated domestic wastewater in four different irrigation configurations: open spray, spray under plastic sheet cover, open drip and drip under plastic sheet cover. Samples of lettuce from each irrigation configuration and irrigating wastewater were collected during the growing season. No E. coli was detected in lettuce from drip irrigated beds. All lettuce samples from spray beds were positive for E. coli, however, no statistical difference (p > 0.05) was detected between lettuces grown in open spray or covered spray beds. The results from the field experiment were also compared to a laboratory experiment which used submersion of lettuce in wastewater of known E. coli concentration as a surrogate method to assess contamination following irrigation. The microbial quality of spray bed lettuces was not significantly different from submersed lettuce when irrigated with wastewater containing 1,299.7 E. coli MPN/100 mL (p > 0.05). This study is significant since it is the first to validate that the microbial contamination of lettuce irrigated with wastewater in the field is comparable with a laboratory technique frequently applied in the quantitative microbial risk assessment of the consumption of wastewater irrigated salad crops.

Detection and Enumeration of Microorganisms in Water and Waste Waters.

ERIC Educational Resources Information Center

Andrews, S.

1980-01-01

Presents and explains analytical procedures for the detection and quantitative enumeration of bacteria which constitute or are indicators of fecal contamination of aquatic environments. Tests are given for Escherichia coli, fecal Streptococci, Clostridium perfringens, and Salmonella. (WB)

A rapid detection method of Escherichia coli by surface enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Tao, Feifei; Peng, Yankun; Xu, Tianfeng

2015-05-01

Conventional microbiological detection and enumeration methods are time-consuming, labor-intensive, and giving retrospective information. The objectives of the present work are to study the capability of surface enhanced Raman scattering (SERS) to detect Escherichia coli (E. coli) using the presented silver colloidal substrate. The obtained results showed that the adaptive iteratively reweighed Penalized Least Squares (airPLS) algorithm could effectively remove the fluorescent background from original Raman spectra, and Raman characteristic peaks of 558, 682, 726, 1128, 1210 and 1328 cm-1 could be observed stably in the baseline corrected SERS spectra of all studied bacterial concentrations. The detection limit of SERS could be determined to be as low as 0.73 log CFU/ml for E. coli with the prepared silver colloidal substrate. The quantitative prediction results using the intensity values of characteristic peaks were not good, with the correlation coefficients of calibration set and cross validation set of 0.99 and 0.64, respectively.

Integrated stoichiometric, thermodynamic and kinetic modelling of steady state metabolism

PubMed Central

Fleming, R.M.T.; Thiele, I.; Provan, G.; Nasheuer, H.P.

2010-01-01

The quantitative analysis of biochemical reactions and metabolites is at frontier of biological sciences. The recent availability of high-throughput technology data sets in biology has paved the way for new modelling approaches at various levels of complexity including the metabolome of a cell or an organism. Understanding the metabolism of a single cell and multi-cell organism will provide the knowledge for the rational design of growth conditions to produce commercially valuable reagents in biotechnology. Here, we demonstrate how equations representing steady state mass conservation, energy conservation, the second law of thermodynamics, and reversible enzyme kinetics can be formulated as a single system of linear equalities and inequalities, in addition to linear equalities on exponential variables. Even though the feasible set is non-convex, the reformulation is exact and amenable to large-scale numerical analysis, a prerequisite for computationally feasible genome scale modelling. Integrating flux, concentration and kinetic variables in a unified constraint-based formulation is aimed at increasing the quantitative predictive capacity of flux balance analysis. Incorporation of experimental and theoretical bounds on thermodynamic and kinetic variables ensures that the predicted steady state fluxes are both thermodynamically and biochemically feasible. The resulting in silico predictions are tested against fluxomic data for central metabolism in E. coli and compare favourably with in silico prediction by flux balance analysis. PMID:20230840

Identification and differentiation of Campylobacter species by high-resolution melting curve analysis.

PubMed

Hoseinpour, Fatemeh; Foroughi, Azadeh; Nomanpour, Bizhan; Nasab, Rezvan Sobhani

2017-07-01

Campylobacter jejuni and Campylobacter coli are the important food-born zoonotic pathogen, also are leading causes of human food borne illnesses worldwide. cadF gene is expressed in all C. jejuni and C. coli strains and mediates cell binding to the cell matrix protein, Fibronectin. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The goal of this study was to apply HRM analysis to identification of C. jejuni and C. coli. A total of 100 samples were obtained from chicken in Kermanshah, Iran. HRM analysis based on cadF gene and Eva Green was developed to the identification of Campylobacter to the species level. Fifty-five of 100 samples were positive as campylobacter (7 C. jejuni, 29 C. coli, 16 mixes and 3 unknown). Minor variations were observed in melting point temperatures of C. coli or C. jejuni isolates and this variation can be used to the differentiation between C. coli or C. jejuni isolates. The results of this study indicated that HRM curve analysis can be a suitable technique and rapid technique for distinguishing between C. jejuni and C. coli isolates. Copyright © 2017 Elsevier Ltd. All rights reserved.

High-performance single cell genetic analysis using microfluidic emulsion generator arrays.

PubMed

Zeng, Yong; Novak, Richard; Shuga, Joe; Smith, Martyn T; Mathies, Richard A

2010-04-15

High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex polymerase chain reaction (PCR). Microfabricated emulsion generator array (MEGA) devices containing 4, 32, and 96 channels are developed to confer a flexible capability of generating up to 3.4 x 10(6) nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed and the beads are pooled and rapidly analyzed by multicolor flow cytometry. Using Escherichia coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1/10(5). This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations.

High-Performance Single Cell Genetic Analysis Using Microfluidic Emulsion Generator Arrays

PubMed Central

Zeng, Yong; Novak, Richard; Shuga, Joe; Smith, Martyn T.; Mathies, Richard A.

2010-01-01

High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex PCR. Microfabricated emulsion generator array (MEGA) devices containing 4, 32 and 96 channels are developed to confer a flexible capability of generating up to 3.4 × 106 nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed, the beads are pooled and rapidly analyzed by multi-color flow cytometry. Using E. coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1:105. This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations. PMID:20192178

Comparison of time-gated surface-enhanced Raman spectroscopy (TG-SERS) and classical SERS based monitoring of Escherichia coli cultivation samples.

PubMed

Kögler, Martin; Paul, Andrea; Anane, Emmanuel; Birkholz, Mario; Bunker, Alex; Viitala, Tapani; Maiwald, Michael; Junne, Stefan; Neubauer, Peter

2018-06-08

The application of Raman spectroscopy as a monitoring technique for bioprocesses is severely limited by a large background signal originating from fluorescing compounds in the culture media. Here we compare time-gated Raman (TG-Raman)-, continuous wave NIR-process Raman (NIR-Raman) and continuous wave micro-Raman (micro-Raman) approaches in combination with surface enhanced Raman spectroscopy (SERS) for their potential to overcome this limit. For that purpose, we monitored metabolite concentrations of Escherichia coli bioreactor cultivations in cell-free supernatant samples. We investigated concentration transients of glucose, acetate, AMP and cAMP at alternating substrate availability, from deficiency to excess. Raman and SERS signals were compared to off-line metabolite analysis of carbohydrates, carboxylic acids and nucleotides. Results demonstrate that SERS, in almost all cases, led to a higher number of identifiable signals and better resolved spectra. Spectra derived from the TG-Raman were comparable to those of micro-Raman resulting in well-discernable Raman peaks, which allowed for the identification of a higher number of compounds. In contrast, NIR-Raman provided a superior performance for the quantitative evaluation of analytes, both with and without SERS nanoparticles when using multivariate data analysis. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

Metabolic Flux Between Unsaturated and Saturated Fatty Acids is Controlled by the FabA:FabB Ratio in the Fully Reconstituted Fatty Acid Biosynthetic Pathway of E. coli#

PubMed Central

Xiao, Xirui; Yu, Xingye; Khosla, Chaitan

2013-01-01

The entire fatty acid biosynthetic pathway from Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from fourteen purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H into the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multi-enzyme system. At steady state, a maximum turnover rate of 0.5 s−1 was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. By altering these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximum turnover rate of the pathway. Our reconstituted system provides a powerful tool to understand and engineer rate-limiting and regulatory steps in this complex and practically significant metabolic pathway. PMID:24147979

Multicenter evaluation of molecular and culture-dependent diagnostics for Shigella species and Entero-invasive Escherichia coli in the Netherlands.

PubMed

van den Beld, Maaike J C; Friedrich, Alexander W; van Zanten, Evert; Reubsaet, Frans A G; Kooistra-Smid, Mirjam A M D; Rossen, John W A

2016-12-01

An inter-laboratory collaborative trial for the evaluation of diagnostics for detection and identification of Shigella species and Entero-invasive Escherichia coli (EIEC) was performed. Sixteen Medical Microbiological Laboratories (MMLs) participated. MMLs were interviewed about their diagnostic methods and a sample panel, consisting of DNA-extracts and spiked stool samples with different concentrations of Shigella flexneri, was provided to each MML. The results of the trial showed an enormous variety in culture-dependent and molecular diagnostic techniques currently used among MMLs. Despite the various molecular procedures, 15 out of 16 MMLs were able to detect Shigella species or EIEC in all the samples provided, showing that the diversity of methods has no effect on the qualitative detection of Shigella flexneri. In contrast to semi quantitative analysis, the minimum and maximum values per sample differed by approximately five threshold cycles (Ct-value) between the MMLs included in the study. This indicates that defining a uniform Ct-value cut-off for notification to health authorities is not advisable. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantifying Impact of Chromosome Copy Number on Recombination in Escherichia coli.

PubMed

Reynolds, T Steele; Gill, Ryan T

2015-07-17

The ability to precisely and efficiently recombineer synthetic DNA into organisms of interest in a quantitative manner is a key requirement in genome engineering. Even though considerable effort has gone into the characterization of recombination in Escherichia coli, there is still substantial variability in reported recombination efficiencies. We hypothesized that this observed variability could, in part, be explained by the variability in chromosome copy number as well as the location of the replication forks relative to the recombination site. During rapid growth, E. coli cells may contain several pairs of open replication forks. While recombineered forks are resolving and segregating within the population, changes in apparent recombineering efficiency should be observed. In the case of dominant phenotypes, we predicted and then experimentally confirmed that the apparent recombination efficiency declined during recovery until complete segregation of recombineered and wild-type genomes had occurred. We observed the reverse trend for recessive phenotypes. The observed changes in apparent recombination efficiency were found to be in agreement with mathematical calculations based on our proposed mechanism. We also provide a model that can be used to estimate the total segregated recombination efficiency based on an initial efficiency and growth rate. These results emphasize the importance of employing quantitative strategies in the design of genome-scale engineering efforts.

Quantitative Microbial Risk Assessment for Escherichia coli O157:H7 in Fresh-Cut Lettuce.

PubMed

Pang, Hao; Lambertini, Elisabetta; Buchanan, Robert L; Schaffner, Donald W; Pradhan, Abani K

2017-02-01

Leafy green vegetables, including lettuce, are recognized as potential vehicles for foodborne pathogens such as Escherichia coli O157:H7. Fresh-cut lettuce is potentially at high risk of causing foodborne illnesses, as it is generally consumed without cooking. Quantitative microbial risk assessments (QMRAs) are gaining more attention as an effective tool to assess and control potential risks associated with foodborne pathogens. This study developed a QMRA model for E. coli O157:H7 in fresh-cut lettuce and evaluated the effects of different potential intervention strategies on the reduction of public health risks. The fresh-cut lettuce production and supply chain was modeled from field production, with both irrigation water and soil as initial contamination sources, to consumption at home. The baseline model (with no interventions) predicted a mean probability of 1 illness per 10 million servings and a mean of 2,160 illness cases per year in the United States. All intervention strategies evaluated (chlorine, ultrasound and organic acid, irradiation, bacteriophage, and consumer washing) significantly reduced the estimated mean number of illness cases when compared with the baseline model prediction (from 11.4- to 17.9-fold reduction). Sensitivity analyses indicated that retail and home storage temperature were the most important factors affecting the predicted number of illness cases. The developed QMRA model provided a framework for estimating risk associated with consumption of E. coli O157:H7-contaminated fresh-cut lettuce and can guide the evaluation and development of intervention strategies aimed at reducing such risk.

Diagnostic accuracy of semi-quantitative and quantitative culture techniques for the diagnosis of catheter-related infections in newborns and molecular typing of isolated microorganisms.

PubMed

Riboli, Danilo Flávio Moraes; Lyra, João César; Silva, Eliane Pessoa; Valadão, Luisa Leite; Bentlin, Maria Regina; Corrente, José Eduardo; Rugolo, Ligia Maria Suppo de Souza; da Cunha, Maria de Lourdes Ribeiro de Souza

2014-05-22

Catheter-related bloodstream infections (CR-BSIs) have become the most common cause of healthcare-associated bloodstream infections in neonatal intensive care units (ICUs). Microbiological evidence implicating catheters as the source of bloodstream infection is necessary to establish the diagnosis of CR-BSIs. Semi-quantitative culture is used to determine the presence of microorganisms on the external catheter surface, whereas quantitative culture also isolates microorganisms present inside the catheter. The main objective of this study was to determine the sensitivity and specificity of these two techniques for the diagnosis of CR-BSIs in newborns from a neonatal ICU. In addition, PFGE was used for similarity analysis of the microorganisms isolated from catheters and blood cultures. Semi-quantitative and quantitative methods were used for the culture of catheter tips obtained from newborns. Strains isolated from catheter tips and blood cultures which exhibited the same antimicrobial susceptibility profile were included in the study as positive cases of CR-BSI. PFGE of the microorganisms isolated from catheters and blood cultures was performed for similarity analysis and detection of clones in the ICU. A total of 584 catheter tips from 399 patients seen between November 2005 and June 2012 were analyzed. Twenty-nine cases of CR-BSI were confirmed. Coagulase-negative staphylococci (CoNS) were the most frequently isolated microorganisms, including S. epidermidis as the most prevalent species (65.5%), followed by S. haemolyticus (10.3%), yeasts (10.3%), K. pneumoniae (6.9%), S. aureus (3.4%), and E. coli (3.4%). The sensitivity of the semi-quantitative and quantitative techniques was 72.7% and 59.3%, respectively, and specificity was 95.7% and 94.4%. The diagnosis of CR-BSIs based on PFGE analysis of similarity between strains isolated from catheter tips and blood cultures showed 82.6% sensitivity and 100% specificity. The semi-quantitative culture method showed higher sensitivity and specificity for the diagnosis of CR-BSIs in newborns when compared to the quantitative technique. In addition, this method is easier to perform and shows better agreement with the gold standard, and should therefore be recommended for routine clinical laboratory use. PFGE may contribute to the control of CR-BSIs by identifying clusters of microorganisms in neonatal ICUs, providing a means of determining potential cross-infection between patients.

Quantitative analysis of commensal Escherichia coli populations reveals host-specific enterotypes at the intra-species level.

PubMed

Smati, Mounira; Clermont, Olivier; Bleibtreu, Alexandre; Fourreau, Frédéric; David, Anthony; Daubié, Anne-Sophie; Hignard, Cécile; Loison, Odile; Picard, Bertrand; Denamur, Erick

2015-08-01

The primary habitat of the Escherichia coli species is the gut of warm-blooded vertebrates. The E. coli species is structured into four main phylogenetic groups A, B1, B2, and D. We estimated the relative proportions of these phylogroups in the feces of 137 wild and domesticated animals with various diets living in the Ile de France (Paris) region by real-time PCR. We distinguished three main clusters characterized by a particular abundance of two or more phylogroups within the E. coli animal commensal populations, which we called "enterocolitypes" by analogy with the enterotypes defined in the human gut microbiota at the genus level. These enterocolitypes were characterized by a dominant (>50%) B2, B1, or A phylogroup and were associated with different host species, diets, and habitats: wild and herbivorous species (wild rabbits and deer), domesticated herbivorous species (domesticated rabbits, horses, sheep, and cows), and omnivorous species (boar, pigs, and chickens), respectively. By analyzing retrospectively the data obtained using the same approach from 98 healthy humans living in Ile de France (Smati et al. 2013, Appl. Environ. Microbiol. 79, 5005-5012), we identified a specific human enterocolitype characterized by the dominant and/or exclusive (>90%) presence of phylogroup B2. We then compared B2 strains isolated from animals and humans, and revealed that human and animal strains differ regarding O-type and B2 subgroup. Moreover, two genes, sfa/foc and clbQ, were associated with the exclusive character of strains, observed only in humans. In conclusion, a complex network of interactions exists at several levels (genus and intra-species) within the intestinal microbiota. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Development of a relative risk model for drinking water regulation and design recommendations for a peri urban region of Argentina.

PubMed

Rodriguez-Alvarez, María Soledad; Weir, Mark H; Pope, Joanna M; Seghezzo, Lucas; Rajal, Verónica B; Salusso, María Mónica; Moraña, Liliana B

2015-10-01

Argentina is a developing Latin American nation that has an aim of achieving the United Nations Millennium Development Goals for potable water supplies. Their current regulations however, limit the continued development of improved potable water quality and infrastructure from a microbiological viewpoint. This is since the current regulations are focused solely to pathogenic Eschericia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and fecal indicators. Regions of lower socioeconomic status such as peri-urban areas are particularly at risk due to lessened financial and political ability to influence their environmental quality and infrastructure needs. Therefore, a combined microbiological sampling, analysis and quantitative microbial risk assessment (QMRA) modeling effort were engaged for a peri-urban area of Salta Argentina. Drinking water samples from home taps were analyzed and a QMRA model was developed, results of which were compared against a general 1:10,000 risk level for lack of a current Argentinian standard. This QMRA model was able to demonstrate that the current regulations were being achieved for E. coli but were less than acceptable for P. aeruginosa in some instances. Appropriate health protections are far from acceptable for Giardia for almost all water sources. Untreated water sources were sampled and analyzed then QMRA modeled as well, since a significant number of the community (∼9%) still use them for potable water supplies. For untreated water E. coli risks were near 1:10,000, however, P. aeruginosa and Giardia risks failed to be acceptable in almost all instances. The QMRA model and microbiological analyses demonstrate the need for improved regulatory efforts for the peri-urban area along with improved investment in their water infrastructure. Copyright © 2015 Elsevier GmbH. All rights reserved.

[Changes of biological behavioral of E. coli K1 after ppk1 gene deletion].

PubMed

Peng, Liang; Pan, Jiayun; Luo, Su; Yang, Zhenghui; Huang, Mufang; Cao, Hong

2014-06-01

To study the changes in biological behaviors of meningitis E. coli K1 strain E44 after deletion of polyphosphate kinase 1 (ppk1) gene and explore the role of ppk1 in the pathogenesis of E. coli K1-induced meningitis. The wild-type strain E. coli K1 and ppk1 deletion mutant were exposed to heat at 56 degrees celsius; for 6 min, and their survival rates were determined. The adhesion and invasion of the bacteria to human brain microvascular endothelial cells (HBMECs) were observed using electron microscopy and quantitative tests. HBMECs were co-incubated with wild-type strain or ppk1 deletion mutant, and the cytoskeleton rearrangement was observed under laser scanning confocal microscope. The survival rate of the ppk1 deletion mutant was significantly lower than that of the wild-type strain after heat exposure. The ppk1 deletion mutant also showed lowered cell adhesion and invasion abilities and weakened ability to induce cytoskeleton rearrangement in HBMECs. ppk1 gene is important for E.coli K1 for heat resistance, cell adhesion and invasion, and for inducing cytoskeletal rearrangement in HBMECs.

Multi-locus variable number tandem repeat analysis for Escherichia coli causing extraintestinal infections.

PubMed

Manges, Amee R; Tellis, Patricia A; Vincent, Caroline; Lifeso, Kimberley; Geneau, Geneviève; Reid-Smith, Richard J; Boerlin, Patrick

2009-11-01

Discriminatory genotyping methods for the analysis of Escherichia coli other than O157:H7 are necessary for public health-related activities. A new multi-locus variable number tandem repeat analysis protocol is presented; this method achieves an index of discrimination of 99.5% and is reproducible and valid when tested on a collection of 836 diverse E. coli.

From qualitative data to quantitative models: analysis of the phage shock protein stress response in Escherichia coli

PubMed Central

2011-01-01

Background Bacteria have evolved a rich set of mechanisms for sensing and adapting to adverse conditions in their environment. These are crucial for their survival, which requires them to react to extracellular stresses such as heat shock, ethanol treatment or phage infection. Here we focus on studying the phage shock protein (Psp) stress response in Escherichia coli induced by a phage infection or other damage to the bacterial membrane. This system has not yet been theoretically modelled or analysed in silico. Results We develop a model of the Psp response system, and illustrate how such models can be constructed and analyzed in light of available sparse and qualitative information in order to generate novel biological hypotheses about their dynamical behaviour. We analyze this model using tools from Petri-net theory and study its dynamical range that is consistent with currently available knowledge by conditioning model parameters on the available data in an approximate Bayesian computation (ABC) framework. Within this ABC approach we analyze stochastic and deterministic dynamics. This analysis allows us to identify different types of behaviour and these mechanistic insights can in turn be used to design new, more detailed and time-resolved experiments. Conclusions We have developed the first mechanistic model of the Psp response in E. coli. This model allows us to predict the possible qualitative stochastic and deterministic dynamic behaviours of key molecular players in the stress response. Our inferential approach can be applied to stress response and signalling systems more generally: in the ABC framework we can condition mathematical models on qualitative data in order to delimit e.g. parameter ranges or the qualitative system dynamics in light of available end-point or qualitative information. PMID:21569396

Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol

PubMed Central

2017-01-01

Tight and tunable control of gene expression is a highly desirable goal in synthetic biology for constructing predictable gene circuits and achieving preferred phenotypes. Elucidating the sequence–function relationship of promoters is crucial for manipulating gene expression at the transcriptional level, particularly for inducible systems dependent on transcriptional regulators. Sort-seq methods employing fluorescence-activated cell sorting (FACS) and high-throughput sequencing allow for the quantitative analysis of sequence–function relationships in a robust and rapid way. Here we utilized a massively parallel sort-seq approach to analyze the formaldehyde-inducible Escherichia coli promoter (Pfrm) with single-nucleotide resolution. A library of mutated formaldehyde-inducible promoters was cloned upstream of gfp on a plasmid. The library was partitioned into bins via FACS on the basis of green fluorescent protein (GFP) expression level, and mutated promoters falling into each expression bin were identified with high-throughput sequencing. The resulting analysis identified two 19 base pair repressor binding sites, one upstream of the −35 RNA polymerase (RNAP) binding site and one overlapping with the −10 site, and assessed the relative importance of each position and base therein. Key mutations were identified for tuning expression levels and were used to engineer formaldehyde-inducible promoters with predictable activities. Engineered variants demonstrated up to 14-fold lower basal expression, 13-fold higher induced expression, and a 3.6-fold stronger response as indicated by relative dynamic range. Finally, an engineered formaldehyde-inducible promoter was employed to drive the expression of heterologous methanol assimilation genes and achieved increased biomass levels on methanol, a non-native substrate of E. coli. PMID:28463494

Quantitative contamination and transfer of Escherichia coli from foods by houseflies, Musca domestica L. (Diptera: Muscidae).

PubMed

De Jesús, Antonio J; Olsen, Alan R; Bryce, John R; Whiting, Richard C

2004-06-01

The housefly, Musca domestica L. (Diptera: Muscidae), is recognized as an important factor in the dissemination of various infectious diseases such as cholera, shigellosis, and salmonellosis. They can also serve as a cross-contamination vector for other foodborne pathogens. However, the potential for bacterial transfer by houseflies has been demonstrated in a qualitative rather than quantitative manner. In this study, the numbers of bacteria a housefly can carry on its body and transfer to a clean surface after exposure to a sugar-milk aqueous solution, steak, and potato salad contaminated with a fluorescent gene Escherichia coli (8 log10 CFU/ml) were determined. In the first series of experiments to quantify bacterial numbers on the flies, about 40-60 flies were transferred into a sterile cage, exposed to the food for 30 min, the flies immobilized and the attached E. coli on each fly enumerated. Detectable E. coli (>1.7 log10 CFU/fly) were found on 43% (29/67), 53% (23/43), and 62% (32/52) of the flies in the cages with sugar/milk, steak, and potato salad, respectively. For the positive flies, the geometric mean carriage (log10 CFU/fly) was 2.93+/-1.24 for sugar-milk, 3.77+/-1.28 for steak, and 2.25+/-0.64 for the potato salad. In the second series of experiments, the transfer of bacteria by individual flies from contaminated food to the inner surface of a sterile jar per each landing was determined. E. coli transferred from the sugar-milk was 3.5+/-0.7 log10 CFU/fly-landing, 3.9+/-0.7 for steak and 2.61+/-1.16 for the potato salad. From the initial contamination levels of bacteria and the number of transferred bacteria, it can be calculated that flies contaminate clean surfaces with approximately 0.1 mg of food per landing. Copyright 2004 Elsevier B.V.

Semi-quantitative evaluation of fecal contamination potential by human and ruminant sources using multiple lines of evidence.

PubMed

Stoeckel, Donald M; Stelzer, Erin A; Stogner, Robert W; Mau, David P

2011-05-01

Protocols for microbial source tracking of fecal contamination generally are able to identify when a source of contamination is present, but thus far have been unable to evaluate what portion of fecal-indicator bacteria (FIB) came from various sources. A mathematical approach to estimate relative amounts of FIB, such as Escherichia coli, from various sources based on the concentration and distribution of microbial source tracking markers in feces was developed. The approach was tested using dilute fecal suspensions, then applied as part of an analytical suite to a contaminated headwater stream in the Rocky Mountains (Upper Fountain Creek, Colorado). In one single-source fecal suspension, a source that was not present could not be excluded because of incomplete marker specificity; however, human and ruminant sources were detected whenever they were present. In the mixed-feces suspension (pet and human), the minority contributor (human) was detected at a concentration low enough to preclude human contamination as the dominant source of E. coli to the sample. Without the semi-quantitative approach described, simple detects of human-associated marker in stream samples would have provided inaccurate evidence that human contamination was a major source of E. coli to the stream. In samples from Upper Fountain Creek the pattern of E. coli, general and host-associated microbial source tracking markers, nutrients, and wastewater-associated chemical detections--augmented with local observations and land-use patterns--indicated that, contrary to expectations, birds rather than humans or ruminants were the predominant source of fecal contamination to Upper Fountain Creek. This new approach to E. coli allocation, validated by a controlled study and tested by application in a relatively simple setting, represents a widely applicable step forward in the field of microbial source tracking of fecal contamination. Copyright © 2011 Elsevier Ltd. All rights reserved.

Escherichia coli K1 induces IL-8 expression in human brain microvascular endothelial cells.

PubMed

Galanakis, Emmanouil; Di Cello, Francescopaolo; Paul-Satyaseela, Maneesh; Kim, Kwang Sik

2006-12-01

Microbial penetration of the blood-brain barrier (BBB) into the central nervous system is essential for the development of meningitis. Considerable progress has been achieved in understanding the pathophysiology of meningitis, however, relatively little is known about the early inflammatory events occurring at the time of bacterial crossing of the BBB. We investigated, using real-time quantitative PCR, the expression of the neutrophil chemoattractants alpha-chemokines CXCL1 (Groalpha) and CXCL8 (IL-8), and of the monocyte chemoattractant beta-chemokine CCL2 (MCP-1) by human brain microvascular endothelial cells (HBMEC) in response to the meningitis-causing E. coli K1 strain RS218 or its isogenic mutants lacking the ability to bind to and invade HBMEC. A nonpathogenic, laboratory E. coli strain HB101 was used as a negative control. CXCL8 was shown to be significantly expressed in HBMEC 4 hours after infection with E. coli K1, while no significant alterations were noted for CXCL1 and CCL2 expression. This upregulation of CXCL8 was induced by E. coli K1 strain RS218 and its derivatives lacking the ability to bind and invade HBMEC, but was not induced by the laboratory strain HB101. In contrast, no upregulation of CXCL8 was observed in human umbilical vein endothelial cells (HUVEC) after stimulation with E. coli RS218. These findings indicate that the CXCL8 expression is the result of the specific response of HBMEC to meningitis-causing E. coli K1.

Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity.

PubMed

Mulligan, M E; Hawley, D K; Entriken, R; McClure, W R

1984-01-11

We describe a simple algorithm for computing a homology score for Escherichia coli promoters based on DNA sequence alone. The homology score was related to 31 values, measured in vitro, of RNA polymerase selectivity, which we define as the product KBk2, the apparent second order rate constant for open complex formation. We found that promoter strength could be predicted to within a factor of +/-4.1 in KBk2 over a range of 10(4) in the same parameter. The quantitative evaluation was linked to an automated (Apple II) procedure for searching and evaluating possible promoters in DNA sequence files.

Characteristics and expression patterns of the aldehyde dehydrogenase (ALDH) gene superfamily of foxtail millet (Setaria italica L.).

PubMed

Chen, Zhu; Chen, Ming; Xu, Zhao-shi; Li, Lian-cheng; Chen, Xue-ping; Ma, You-zhi

2014-01-01

Recent genomic sequencing of the foxtail millet, an abiotic, stress-tolerant crop, has provided a great opportunity for novel gene discovery and functional analysis of this popularly-grown grass. However, few stress-mediated gene families have been studied. Aldehyde dehydrogenases (ALDHs) comprise a gene superfamily encoding NAD (P) +-dependent enzymes that play the role of "aldehyde scavengers", which indirectly detoxify cellular ROS and reduce the effect of lipid peroxidation meditated cellular toxicity under various environmental stresses. In the current paper, we identified a total of 20 ALDH genes in the foxtail millet genome using a homology search and a phylogenetic analysis and grouped them into ten distinct families based on their amino acid sequence identity. Furthermore, evolutionary analysis of foxtail millet reveals that both tandem and segmental duplication contributed significantly to the expansion of its ALDH genes. The exon-intron structures of members of the same family in foxtail millet or the orthologous genes in rice display highly diverse distributions of their exonic and intronic regions. Also, synteny analysis shows that the majority of foxtail millet and rice ALDH gene homologs exist in the syntenic blocks between the two, implying that these ALDH genes arose before the divergence of cereals. Semi-quantitative and real-time quantitative PCR data reveals that a few SiALDH genes are expressed in an organ-specific manner and that the expression of a number of foxtail millet ALDH genes, such as, SiALDH7B1, SiALDH12A1 and SiALDH18B2 are up-regulated by osmotic stress, cold, H2O2, and phytohormone abscisic acid (ABA). Furthermore, the transformation of SiALDH2B2, SiALDH10A2, SiALDH5F1, SiALDH22A1, and SiALDH3E2 into Escherichia coli (E.coli) was able to improve their salt tolerance. Taken together, our results show that genome-wide identification characteristics and expression analyses provide unique opportunities for assessing the functional roles of foxtail millet ALDH genes in stress responses.

Uncertainty in monitoring E. coli concentrations in streams and stormwater runoff

NASA Astrophysics Data System (ADS)

Harmel, R. D.; Hathaway, J. M.; Wagner, K. L.; Wolfe, J. E.; Karthikeyan, R.; Francesconi, W.; McCarthy, D. T.

2016-03-01

Microbial contamination of surface waters, a substantial public health concern throughout the world, is typically identified by fecal indicator bacteria such as Escherichia coli. Thus, monitoring E. coli concentrations is critical to evaluate current conditions, determine restoration effectiveness, and inform model development and calibration. An often overlooked component of these monitoring and modeling activities is understanding the inherent random and systematic uncertainty present in measured data. In this research, a review and subsequent analysis was performed to identify, document, and analyze measurement uncertainty of E. coli data collected in stream flow and stormwater runoff as individual discrete samples or throughout a single runoff event. Data on the uncertainty contributed by sample collection, sample preservation/storage, and laboratory analysis in measured E. coli concentrations were compiled and analyzed, and differences in sampling method and data quality scenarios were compared. The analysis showed that: (1) manual integrated sampling produced the lowest random and systematic uncertainty in individual samples, but automated sampling typically produced the lowest uncertainty when sampling throughout runoff events; (2) sample collection procedures often contributed the highest amount of uncertainty, although laboratory analysis introduced substantial random uncertainty and preservation/storage introduced substantial systematic uncertainty under some scenarios; and (3) the uncertainty in measured E. coli concentrations was greater than that of sediment and nutrients, but the difference was not as great as may be assumed. This comprehensive analysis of uncertainty in E. coli concentrations measured in streamflow and runoff should provide valuable insight for designing E. coli monitoring projects, reducing uncertainty in quality assurance efforts, regulatory and policy decision making, and fate and transport modeling.

[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.

A double-taper optical fiber-based radiation wave other than evanescent wave in all-fiber immunofluorescence biosensor for quantitative detection of Escherichia coli O157:H7.

PubMed

Zhang, Zhonghuan; Hua, Fei; Liu, Ting; Zhao, Yong; Li, Jun; Yang, Ruifu; Yang, Changxi; Zhou, Lei

2014-01-01

Cylindrical or taper-and-cylinder combination optical fiber probe based on evanescent wave has been widely used for immunofluorescence biosensor to detect various analytes. In this study, in contrast to the contradiction between penetration depth and analyte diameter of optical fiber probe-based evanescent wave, we demonstrate that double-taper optical fiber used in a radiation wave-based all-fiber immunofluorescence biosensor (RWAIB) can detect micron-scale analytes using Escherichia coli O157:H7 as representative target. Finite-difference time-domain method was used to compare the properties of evanescent wave and radiation wave (RW). Ray-tracing model was formulated to optimize the taper geometry of the probe. Based on a commercial multi-mode fiber, a double-taper probe was fabricated and connected with biosensor through a "ferrule connector" optical fiber connector. The RWAIB configuration was accomplished using commercial multi-mode fibers and fiber-based devices according to the "all-fiber" method. The standard sample tests revealed that the sensitivity of the proposed technique for E. coli O157:H7 detection was 10(3) cfu · mL(-1). Quantitation could be achieved within the concentration range of 10(3) cfu · mL(-1) to 107 cfu · mL(-1). No non-specific recognition to ten kinds of food-borne pathogens was observed. The results demonstrated that based on the double-taper optical fiber RWAIB can be used for the quantitative detection of micron-scale targets, and RW sensing is an alternative for traditional evanescent wave sensing during the fabrication of fiber-optic biosensors.

Evaluation and single-laboratory verification of a proposed modification to the U.S. Food and Drug Administration method for detection and identification of Campylobacter jejuni or Campylobacter coli from raw silo milk.

PubMed

Gharst, Greg; Bark, Don H; Newkirk, Robert; Guillen, Lacey; Wang, Qian; Abeyta, Carlos

2013-01-01

The current U.S. Food and Drug Administration (FDA) methodology for detection of Campylobacter, a leading source for foodborne illness, is outdated. The purpose of this study, therefore, was to improve and update the cultural and identification methods found in the FDA/Bacteriological Analytical Manual (BAM). Raw silo milk samples containing typical and atypical strains of Campylobacter jejuni and Campylobacter coli at different levels (5 CFU/25 g, 50 CFU/25 g, and 125 CFU/25 g) were analyzed. Valid results were obtained from 240 test portions. Six inoculated (at the levels described above) and two uninoculated samples were sent to a participating laboratory to mimic a "real-world" scenario. These combined data indicated that the use of sheep blood in combination with enrichment is not necessary. R & F Campylobacter jejuni/Campylobacter coli Chromogenic Plating Medium is significantly (P < 0.05) more sensitive for detection of C. jejuni or C. coli at low inoculation levels than the modified Cefoperazone Charcoal Deoxycholate Agar used in the BAM. The quantitative PCR method described demonstrated rapid confirmation and identification of C. jejuni or C. coli. It reduced the time to isolate C. jejuni or C. coli, and increased the sensitivity compared to the current BAM protocol.

Temperature control of fimbriation circuit switch in uropathogenic Escherichia coli: quantitative analysis via automated model abstraction.

PubMed

Kuwahara, Hiroyuki; Myers, Chris J; Samoilov, Michael S

2010-03-26

Uropathogenic Escherichia coli (UPEC) represent the predominant cause of urinary tract infections (UTIs). A key UPEC molecular virulence mechanism is type 1 fimbriae, whose expression is controlled by the orientation of an invertible chromosomal DNA element-the fim switch. Temperature has been shown to act as a major regulator of fim switching behavior and is overall an important indicator as well as functional feature of many urologic diseases, including UPEC host-pathogen interaction dynamics. Given this panoptic physiological role of temperature during UTI progression and notable empirical challenges to its direct in vivo studies, in silico modeling of corresponding biochemical and biophysical mechanisms essential to UPEC pathogenicity may significantly aid our understanding of the underlying disease processes. However, rigorous computational analysis of biological systems, such as fim switch temperature control circuit, has hereto presented a notoriously demanding problem due to both the substantial complexity of the gene regulatory networks involved as well as their often characteristically discrete and stochastic dynamics. To address these issues, we have developed an approach that enables automated multiscale abstraction of biological system descriptions based on reaction kinetics. Implemented as a computational tool, this method has allowed us to efficiently analyze the modular organization and behavior of the E. coli fimbriation switch circuit at different temperature settings, thus facilitating new insights into this mode of UPEC molecular virulence regulation. In particular, our results suggest that, with respect to its role in shutting down fimbriae expression, the primary function of FimB recombinase may be to effect a controlled down-regulation (rather than increase) of the ON-to-OFF fim switching rate via temperature-dependent suppression of competing dynamics mediated by recombinase FimE. Our computational analysis further implies that this down-regulation mechanism could be particularly significant inside the host environment, thus potentially contributing further understanding toward the development of novel therapeutic approaches to UPEC-caused UTIs.

Interaction of the E. coli DNA G:T-mismatch endonuclease (vsr protein) with oligonucleotides containing its target sequence.

PubMed

Turner, D P; Connolly, B A

2000-12-15

The Escherichia coli vsr endonuclease recognises G:T base-pair mismatches in double-stranded DNA and initiates a repair pathway by hydrolysing the phosphate group 5' to the incorrectly paired T. The enzyme shows a preference for G:T mismatches within a particular sequence context, derived from the recognition site of the E. coli dcm DNA-methyltransferase (CC[A/T]GG). Thus, the preferred substrate for the vsr protein is (CT[A/T]GG), where the underlined T is opposed by a dG base. This paper provides quantitative data for the interaction of the vsr protein with a number of oligonucleotides containing G:T mismatches. Evaluation of specificity constant (k(st)/K(D); k(st)=rate constant for single turnover, K(D)=equilibrium dissociation constant) confirms vsr's preference for a G:T mismatch within a hemi-methylated dcm sequence, i.e. the best substrate is a duplex (both strands written in the 5'-3' orientation) composed of CT[A/T]GG and C(5Me)C[T/A]GG. Conversion of the mispaired T (underlined) to dU or the d(5Me)C to dC gave poorer substrates. No interaction was observed with oligonucleotides that lacked a G:T mismatch or did not possess a dcm sequence. An analysis of the fraction of active protein, by "reverse-titration" (i.e. adding increasing amounts of DNA to a fixed amount of protein followed by gel-mobility shift analysis) showed that less than 1% of the vsr endonuclease was able to bind to the substrate. This was confirmed using "competitive titrations" (where competitor oligonucleotides are used to displace a (32)P-labelled nucleic acid from the vsr protein) and burst kinetic analysis. This result is discussed in the light of previous in vitro and in vivo data which indicate that the MutL protein may be needed for full vsr activity. Copyright 2000 Academic Press.

A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity.

PubMed

O'Brien-Simpson, Neil M; Pantarat, Namfon; Attard, Troy J; Walsh, Katrina A; Reynolds, Eric C

2016-01-01

We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy.

Diversity in ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging

PubMed Central

Yaginuma, Hideyuki; Kawai, Shinnosuke; Tabata, Kazuhito V.; Tomiyama, Keisuke; Kakizuka, Akira; Komatsuzaki, Tamiki; Noji, Hiroyuki; Imamura, Hiromi

2014-01-01

Recent advances in quantitative single-cell analysis revealed large diversity in gene expression levels between individual cells, which could affect the physiology and/or fate of each cell. In contrast, for most metabolites, the concentrations were only measureable as ensemble averages of many cells. In living cells, adenosine triphosphate (ATP) is a critically important metabolite that powers many intracellular reactions. Quantitative measurement of the absolute ATP concentration in individual cells has not been achieved because of the lack of reliable methods. In this study, we developed a new genetically-encoded ratiometric fluorescent ATP indicator “QUEEN”, which is composed of a single circularly-permuted fluorescent protein and a bacterial ATP binding protein. Unlike previous FRET-based indicators, QUEEN was apparently insensitive to bacteria growth rate changes. Importantly, intracellular ATP concentrations of numbers of bacterial cells calculated from QUEEN fluorescence were almost equal to those from firefly luciferase assay. Thus, QUEEN is suitable for quantifying the absolute ATP concentration inside bacteria cells. Finally, we found that, even for a genetically-identical Escherichia coli cell population, absolute concentrations of intracellular ATP were significantly diverse between individual cells from the same culture, by imaging QUEEN signals from single cells. PMID:25283467

Comparison and cost analysis of drinking water quality monitoring requirements versus practice in seven developing countries.

PubMed

Crocker, Jonny; Bartram, Jamie

2014-07-18

Drinking water quality monitoring programs aim to support provision of safe drinking water by informing water quality management. Little evidence or guidance exists on best monitoring practices for low resource settings. Lack of financial, human, and technological resources reduce a country's ability to monitor water supply. Monitoring activities were characterized in Cambodia, Colombia, India (three states), Jordan, Peru, South Africa, and Uganda according to water sector responsibilities, monitoring approaches, and marginal cost. The seven study countries were selected to represent a range of low resource settings. The focus was on monitoring of microbiological parameters, such as E. coli, coliforms, and H2S-producing microorganisms. Data collection involved qualitative and quantitative methods. Across seven study countries, few distinct approaches to monitoring were observed, and in all but one country all monitoring relied on fixed laboratories for sample analysis. Compliance with monitoring requirements was highest for operational monitoring of large water supplies in urban areas. Sample transport and labor for sample collection and analysis together constitute approximately 75% of marginal costs, which exclude capital costs. There is potential for substantive optimization of monitoring programs by considering field-based testing and by fundamentally reconsidering monitoring approaches for non-piped supplies. This is the first study to look quantitatively at water quality monitoring practices in multiple developing countries.

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

PubMed Central

Kosek, Margaret N.; Schwab, Kellogg J.

2017-01-01

Empiric quantification of environmental fecal contamination is an important step toward understanding the impact that water, sanitation, and hygiene interventions have on reducing enteric infections. There is a need to standardize the methods used for surface sampling in field studies that examine fecal contamination in low-income settings. The dry cloth method presented in this manuscript improves upon the more commonly used swabbing technique that has been shown in the literature to have a low sampling efficiency. The recovery efficiency of a dry electrostatic cloth sampling method was evaluated using Escherichia coli and then applied to household surfaces in Iquitos, Peru, where there is high fecal contamination and enteric infection. Side-by-side measurements were taken from various floor locations within a household at the same time over a three-month period to compare for consistency of quantification of E. coli bacteria. The dry cloth sampling method in the laboratory setting showed 105% (95% Confidence Interval: 98%, 113%) E. coli recovery efficiency off of the cloths. The field application demonstrated strong agreement of side-by-side results (Pearson correlation coefficient for dirt surfaces was 0.83 (p < 0.0001) and 0.91 (p < 0.0001) for cement surfaces) and moderate agreement for results between entrance and kitchen samples (Pearson (0.53, p < 0.0001) and weighted Kappa statistic (0.54, p < 0.0001)). Our findings suggest that this method can be utilized in households with high bacterial loads using either continuous (quantitative) or categorical (semi-quantitative) data. The standardization of this low-cost, dry electrostatic cloth sampling method can be used to measure differences between households in intervention and non-intervention arms of randomized trials. PMID:28829392

Development of a rapid and sensitive method combining a cellulose ester microfilter and a real-time quantitative PCR assay to detect Campylobacter jejuni and Campylobacter coli in 20 liters of drinking water or low-turbidity waters.

PubMed

Tissier, Adeline; Denis, Martine; Hartemann, Philippe; Gassilloud, Benoît

2012-02-01

Investigations of Campylobacter jejuni and Campylobacter coli in samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection of Campylobacter in 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (±22% [standard deviation]) of Campylobacter per 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with a C. coli strain were 69.5% (±10.3%) and 78.5% (±15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence of C. jejuni and C. coli genomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination by Campylobacter.

Development of a Rapid and Sensitive Method Combining a Cellulose Ester Microfilter and a Real-Time Quantitative PCR Assay To Detect Campylobacter jejuni and Campylobacter coli in 20 Liters of Drinking Water or Low-Turbidity Waters

PubMed Central

Tissier, Adeline; Denis, Martine; Hartemann, Philippe

2012-01-01

Investigations of Campylobacter jejuni and Campylobacter coli in samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection of Campylobacter in 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (±22% [standard deviation]) of Campylobacter per 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with a C. coli strain were 69.5% (±10.3%) and 78.5% (±15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence of C. jejuni and C. coli genomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination by Campylobacter. PMID:22138985

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

PubMed

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

2017-08-22

Empiric quantification of environmental fecal contamination is an important step toward understanding the impact that water, sanitation, and hygiene interventions have on reducing enteric infections. There is a need to standardize the methods used for surface sampling in field studies that examine fecal contamination in low-income settings. The dry cloth method presented in this manuscript improves upon the more commonly used swabbing technique that has been shown in the literature to have a low sampling efficiency. The recovery efficiency of a dry electrostatic cloth sampling method was evaluated using Escherichia coli and then applied to household surfaces in Iquitos, Peru, where there is high fecal contamination and enteric infection. Side-by-side measurements were taken from various floor locations within a household at the same time over a three-month period to compare for consistency of quantification of E. coli bacteria. The dry cloth sampling method in the laboratory setting showed 105% (95% Confidence Interval: 98%, 113%) E. coli recovery efficiency off of the cloths. The field application demonstrated strong agreement of side-by-side results (Pearson correlation coefficient for dirt surfaces was 0.83 ( p < 0.0001) and 0.91 ( p < 0.0001) for cement surfaces) and moderate agreement for results between entrance and kitchen samples (Pearson (0.53, p < 0.0001) and weighted Kappa statistic (0.54, p < 0.0001)). Our findings suggest that this method can be utilized in households with high bacterial loads using either continuous (quantitative) or categorical (semi-quantitative) data. The standardization of this low-cost, dry electrostatic cloth sampling method can be used to measure differences between households in intervention and non-intervention arms of randomized trials.

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

PubMed

Wang, Shun; Xie, Jiufeng; Jiang, Min; Chang, Keke; Chen, Ruipeng; Ma, Liuzheng; Zhu, Juanhua; Guo, Qingqian; Sun, Haifeng; Hu, Jiandong

2016-11-04

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

Impedimetric detection of bacteria by using a microfluidic chip and silver nanoparticle based signal enhancement.

PubMed

Wang, Renjie; Xu, Yi; Sors, Thomas; Irudayaraj, Joseph; Ren, Wen; Wang, Rong

2018-02-19

The authors describe a method that can significantly improve the performance of impedimetric detection of bacteria. A multifunctional microfluidic chip was designed consisting of interdigitated microelectrodes and a micro-mixing zone with a Tesla structure. This maximizes the coating of bacterial surfaces with nanoparticles and results in improved impedimetric detection. The method was applied to the detection of Escherichia coli O157:H7 (E. coli). Silver enhancement was accomplished by coating E.coli with the cationic polymer diallyldimethylammonium chloride (PDDA) to form positively charged E. coli/PDDA complexes. Then, gold nanoparticles (AuNPs) were added, and the resulting E. coli/PDDA/AuNPs complexes were collected at interdigitated electrodes via positive dielectrophoresis (pDEP). A silver adduct was then formed on the E. coli/PDDA/AuNP complexes by using silver enhancement solutions and by using the AuNPs as catalysts. The combination of pDEP based capture and of using silver adducts reduces impedance by increasing the conductivity of the solution and the double layer capacitance around the microelectrodes. Impedance decreases linearly in the 2 × 10 3 -2 × 10 5  cfu·mL -1 E. coli concentration range, with a 500 cfu·mL -1 detection limit. Egg shell wash samples and tap water spiked with E. coli were successfully used for validation, and this demonstrates the practical application of this method. Graphical abstract Schematic representation of the AuNP@Ag enhancement method integrated with multifunctional microfluidic chip platform for impedimetric quantitation of bacteria. The method significantly improves the performance of impedimetric detection of bacteria.

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

PubMed Central

Wang, Shun; Xie, Jiufeng; Jiang, Min; Chang, Keke; Chen, Ruipeng; Ma, Liuzheng; Zhu, Juanhua; Guo, Qingqian; Sun, Haifeng; Hu, Jiandong

2016-01-01

The purpose of this study was to develop a portable surface plasmon resonance (SPR) bioanalyzer for the sensitive detection of Escherichia coli O157:H7 in comparison with an enzyme-linked immunosorbent assay (ELISA). The experimental setup mainly consisted of an integrated biosensor and a homemade microfluidic cell with a three-way solenoid valve. In order to detect Escherichia coli O157:H7 using the SPR immunoassay, 3-mercaptopropionic acid (3-MPA) was chemisorbed onto a gold surface via covalent bond for the immobilization of biological species. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were used as crosslinker reagents to enable the reaction between 3-MPA and Escherichia coli O157:H7 antibodies by covalent –CO–NH– amide bonding. The experimental results were obtained from the Escherichia coli O157:H7 positive samples prepared by 10-, 20-, 40-, 80-, and 160-fold dilution respectively, which show that a good linear relationship with the correlation coefficient R of 0.982 existed between the response units from the portable SPR bioanalyzer and the concentration of Escherichia coli O157:H7 positive samples. Moreover, the theoretical detection limit of 1.87 × 103 cfu/mL was calculated from the positive control samples. Compared with the Escherichia coli O157:H7 ELISA kit, the sensitivity of this portable SPR bioanalyzer is four orders of magnitude higher than the ELISA kit. The results demonstrate that the portable SPR bioanalyzer could provide an alternative method for the quantitative and sensitive determination of Escherichia coli O157:H7 in field. PMID:27827923

Survival and interaction of Escherichia coli O104:H4 on Arabidopsis thaliana and lettuce (Lactuca sativa) in comparison to E. coli O157:H7: Influence of plant defense response and bacterial capsular polysaccharide.

PubMed

Jang, Hyein; Matthews, Karl R

2018-06-01

Shiga toxin-producing Escherichia coli (STEC) has been associated with illnesses and outbreaks linked to fresh vegetables, prompting a growing public health concern. Most studies regarding interactions of STEC on fresh produce focused on E. coli O157:H7. Limited information is available about survival or fitness of E. coli O104:H4, non-O157 pathogen that was linked to one of the largest outbreaks of hemolytic uremic syndrome in 2011. In this study, survival of E. coli O104:H4 was evaluated on Arabidopsis thaliana plant and lettuce for 5 days compared with E. coli O157:H7, and expression of pathogenesis-realted gene (PR1; induction of plant defense response) was examined by reverse transcription quantitative PCR, and potential influence of capsular polysaccharide (CPS) on the bacterial fitness on plant was investigated. Populations of E. coli O104:H4 strains (RG1, C3493, and LpfA) on Arabidopsis and lettuce were significantly (P < 0.05) greater than those of E. coli O157:H7 strains (7386 and sakai) at day 5 post-inoculation, indicating E. coli O104:H4 may have better survival ability on the plants. In addition, the E. coli O104:H4 strains produced significantly (P < 0.05) higher amounts of CPS compared with the E. coli O157:H7 strains. RG1 strain (1.5-fold) initiated significantly (P < 0.05) lower expression of PR1 gene indicating induction of plant defense response compared with E. coli O157:H7 strains 7386 (2.9-fold) and sakai (2.7-fold). Collectively, the results in this study suggests that different level of CPS production and plant defense response initiated by each STEC strain might influence the bacterial survival or persistence on plants. The present study provides better understanding of survival behavior of STEC, particularly E. coli O104:H4, using a model plant and vegetable under pre-harvest conditions with plant defense response. Copyright © 2018 Elsevier Ltd. All rights reserved.

Detection of viable but non-culturable Escherichia coli O157:H7 by PCR in combination with propidium monoazide.

PubMed

Zhong, Junliang; Zhao, Xihong

2018-01-01

The aim of this study was to evaluate the applicability of the conventional PCR detection method combined with propidium monoazide (PMA) treatment for the detection of viable but non-culturable (VBNC) state Escherichia coli O157:H7 in ground beef meatballs. Under low temperature, E. coli O157:H7 cells were induced into the VBNC state in ground beef meatballs at - 20 °C after 152 days. The optimal PMA concentration of 5 µg/mL was obtained in beef meatball samples, which could completely inhibit the DNA amplification on dead cells (10 6  cells/mL) but with no inhibition on viable cells. The established PMA-PCR assay revealed that the VBNC counts exceeded 10 7  CFU/mL in artificial contamination beef samples, which could be used for semi-quantitative detection of VBNC cells in beef meatball samples. This study indicated that the PMA-PCR assay might be a potential method for detection of VBNC state E . coli O157:H7 cells in food products.

Individual and collective contributions of chaperoning and degradation to protein homeostasis in E. coli.

PubMed

Cho, Younhee; Zhang, Xin; Pobre, Kristine Faye R; Liu, Yu; Powers, David L; Kelly, Jeffery W; Gierasch, Lila M; Powers, Evan T

2015-04-14

The folding fate of a protein in vivo is determined by the interplay between a protein's folding energy landscape and the actions of the proteostasis network, including molecular chaperones and degradation enzymes. The mechanisms of individual components of the E. coli proteostasis network have been studied extensively, but much less is known about how they function as a system. We used an integrated experimental and computational approach to quantitatively analyze the folding outcomes (native folding versus aggregation versus degradation) of three test proteins biosynthesized in E. coli under a variety of conditions. Overexpression of the entire proteostasis network benefited all three test proteins, but the effect of upregulating individual chaperones or the major degradation enzyme, Lon, varied for proteins with different biophysical properties. In sum, the impact of the E. coli proteostasis network is a consequence of concerted action by the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), and Lon. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

[Quantitative study of the antibacterial effect of cefotaxime and ceftriaxone during experimental Escherichia coli K1 bacteremia in chickens].

PubMed

Labarthe, J C; Guillot, J F; Mouline, C; Bree, A

1989-06-01

In order to assess the in vivo antibacterial activity of two cephalosporins of third generation, cefotaxime and ceftriaxone, we used the model of experimental bacteremia in chickens we had developed for a few years. 93 chickens were inoculated with 10(7) E. coli K1 coming from a meningitis in a new-born baby. 19 chickens were used as a control group; 29 were given ceftriaxone (50 mg/kg); 28 cefotaxime (50 mg/kg) and 17 cefotaxime (100 mg/kg). The antibiotics were injected 4 hours after the inoculation. The bacterial concentrations found in capillaries by using quantitative blood cultures, were significantly lower in the 3 groups of chickens which were given antibiotics than in the control group, at 24, 48 and 72 hours after inoculation. At 24 hours after the inoculation, the bacterial concentration in the chickens treated by ceftriaxone (50 mg/kg) was significantly lower than that found in chickens treated by cefotaxime (50 mg/kg). At 48 or 72 hours the differences of bacterial concentration in the three groups of chickens were not significant. Over 72 hours following inoculation, 4 control and only one treated chickens died. The efficient clearance of E. coli K1 by a single dose of ceftriaxone, found at 24 hours after inoculation, confirms the possibility of using ceftriaxone once daily for serious infections.

The infant rat as a model of bacterial meningitis.

PubMed

Moxon, E R; Glode, M P; Sutton, A; Robbins, J B

1977-08-01

The pathogenesis of bacterial meningitis was studied in infant rats. Intranasal intoculation of greater than 10(3) Haemophilus influenzae type b resulted in an incidence of bacteremia that was directly related to the size of hte challenge inoculum. The temporal and quantitative relationship of bacteremia to meningitis indicated that bacteria spread to the meninges by the hematogenous route and that the magnitude of bacteremia was a primary determinant in the development of meningitis. In a sparate series of experiments, infant rats that were fed Escherichia coli strain C94 (O7:K1:H-) became colonized and developed bacteremia and meningitis, but invasive disease was rare when rats were fed E. Coli strain Easter (O75:K100:H5). A comparison of intranasal vs. oral challenge indicated that the nasopharynx was the most effective route for inducing H. influenzae bacteremia, whereas the gastrointestinal route was the more effective challenge route for the E. coli K1 serotype.

Effect of disinfection upon dissolved organic carbon (DOC) in wastewater: bacterial bioassays.

PubMed

Arana, I; Santorum, P; Muela, A; Barcina, I

2000-08-01

Quantitative and qualitative changes in organic matter content of wastewater effluents attributable to chlorination and ozonation have been analysed using bioassays as well as organic carbon direct measures. Bioassays were carried out using the bacterial populations of wastewater and two Escherichia coli strains as test micro-organisms. Our results indicate that pure strains present some advantages over indigenous bacteria. Although wastewater bacterial populations are better adapted to growth in wastewater, E. coli strains are more sensitive to changes in dissolved organic carbon (DOC) content. Moreover, the use of pure cultures allows estimation of the portion of DOC which can be converted in cell biomass, the assimilable organic carbon (AOC). Finally, the results obtained using prototrophic and the auxotrophic strains of E. coli suggested that ozonation alters the amino acid composition of wastewater while chlorination does not change the quantity nor the quality of the DOC present in effluents.

Comparative genomics and transcriptomics of Escherichia coli isolates carrying virulence factors of both enteropathogenic and enterotoxigenic E. coli.

PubMed

Hazen, Tracy H; Michalski, Jane; Luo, Qingwei; Shetty, Amol C; Daugherty, Sean C; Fleckenstein, James M; Rasko, David A

2017-06-14

Escherichia coli that are capable of causing human disease are often classified into pathogenic variants (pathovars) based on their virulence gene content. However, disease-associated hybrid E. coli, containing unique combinations of multiple canonical virulence factors have also been described. Such was the case of the E. coli O104:H4 outbreak in 2011, which caused significant morbidity and mortality. Among the pathovars of diarrheagenic E. coli that cause significant human disease are the enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC). In the current study we use comparative genomics, transcriptomics, and functional studies to characterize isolates that contain virulence factors of both EPEC and ETEC. Based on phylogenomic analysis, these hybrid isolates are more genomically-related to EPEC, but appear to have acquired ETEC virulence genes. Global transcriptional analysis using RNA sequencing, demonstrated that the EPEC and ETEC virulence genes of these hybrid isolates were differentially-expressed under virulence-inducing laboratory conditions, similar to reference isolates. Immunoblot assays further verified that the virulence gene products were produced and that the T3SS effector EspB of EPEC, and heat-labile toxin of ETEC were secreted. These findings document the existence and virulence potential of an E. coli pathovar hybrid that blurs the distinction between E. coli pathovars.

Simultaneous virtual prediction of anti-Escherichia coli activities and ADMET profiles: A chemoinformatic complementary approach for high-throughput screening.

PubMed

Speck-Planche, Alejandro; Cordeiro, M N D S

2014-02-10

Escherichia coli remains one of the principal pathogens that cause nosocomial infections, medical conditions that are increasingly common in healthcare facilities. E. coli is intrinsically resistant to many antibiotics, and multidrug-resistant strains have emerged recently. Chemoinformatics has been a great ally of experimental methodologies such as high-throughput screening, playing an important role in the discovery of effective antibacterial agents. However, there is no approach that can design safer anti-E. coli agents, because of the multifactorial nature and complexity of bacterial diseases and the lack of desirable ADMET (absorption, distribution, metabolism, elimination, and toxicity) profiles as a major cause of disapproval of drugs. In this work, we introduce the first multitasking model based on quantitative-structure biological effect relationships (mtk-QSBER) for simultaneous virtual prediction of anti-E. coli activities and ADMET properties of drugs and/or chemicals under many experimental conditions. The mtk-QSBER model was developed from a large and heterogeneous data set of more than 37800 cases, exhibiting overall accuracies of >95% in both training and prediction (validation) sets. The utility of our mtk-QSBER model was demonstrated by performing virtual prediction of properties for the investigational drug avarofloxacin (AVX) under 260 different experimental conditions. Results converged with the experimental evidence, confirming the remarkable anti-E. coli activities and safety of AVX. Predictions also showed that our mtk-QSBER model can be a promising computational tool for virtual screening of desirable anti-E. coli agents, and this chemoinformatic approach could be extended to the search for safer drugs with defined pharmacological activities.

Comparison of early and late treatment with a recombinant endotoxin neutralizing protein in a rat model of Escherichia coli sepsis.

PubMed

Weiner, D L; Kuppermann, N; Saladino, R A; Thompson, C M; Novitsky, T J; Siber, G R; Fleisher, G R

1996-09-01

To test the efficacy of a recombinant endotoxin neutralizing protein as compared with saline in rats with Escherichia coli sepsis. Prospective, controlled animal trial. Hospital animal research laboratory. Male Wistar rats challenged with intraperitoneal E. coli, O18ac K1, and treated 1 hr later with ceftriaxone and gentamicin. Recombinant endotoxin neutralizing protein, 50 mg/kg, was administered to rats 1, 2, or 3 hrs after E. coli challenge; saline was administered to control animals. Quantitative bacteremia, 1 hr after challenge and before antibiotic administration, was not significantly different between treatment groups (range geometric mean 451 to 621 colony-forming units [cfu]/mL). The endotoxin concentration, measured immediately before recombinant endotoxin neutralizing protein administration, was significantly higher in animals sampled and treated at 2 hrs (geometric mean 260 EU/mL; 95% confidence interval 140 to 480 EU/mL), or 3 hrs (geometric mean 697 EU/mL; 95% confidence interval 307 to 1585 EU/mL) after E. coli challenge, compared with animals sampled and treated at 1 hr (geometric mean 17 EU/mL; 95% confidence interval 7 to 69 EU/ mL). Survival rate was significantly greater in rats treated with recombinant endotoxin neutralizing protein at 1 hr (23/27; p < .001) or 2 hrs (8/30; p < .01) after E. coli challenge than in controls (1/32). Administration of recombinant endotoxin neutralizing protein delayed up to 2 hrs after challenge with E. coli improves survival in antibiotic-treated rats with Gram-negative sepsis.

Distinct Signature of Oxylipid Mediators of Inflammation during Infection and Asymptomatic Colonization by E. coli in the Urinary Bladder

PubMed Central

Packiriswamy, Nandakumar; Gandy, Jeff; Smith, Sara N.; Mobley, Harry L. T.

2017-01-01

Urinary tract infection (UTI) is an extremely common infectious disease. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. Asymptomatic bacteriuric E. coli (ABEC) strains successfully colonize the urinary tract resulting in asymptomatic bacteriuria (ABU) and do not induce symptoms associated with UTI. Oxylipids are key signaling molecules involved in inflammation. Based on the distinct clinical outcomes of E. coli colonization, we hypothesized that UPEC triggers the production of predominantly proinflammatory oxylipids and ABEC leads to production of primarily anti-inflammatory or proresolving oxylipids in the urinary tract. We performed quantitative detection of 39 oxylipid mediators with proinflammatory, anti-inflammatory, and proresolving properties, during UTI and ABU caused by genetically distinct E. coli strains in the murine urinary bladder. Our results reveal that infection with UPEC causes an increased accumulation of proinflammatory oxylipids as early as 6 h postinoculation, compared to controls. To the contrary, ABEC colonization leads to decreased accumulation of proinflammatory oxylipids at the early time point compared to UPEC infection but does not affect the level of proresolving oxylipids. This report represents the first comprehensive investigation on the oxylipidome during benign ABEC colonization observed in ABU and acute inflammation triggered by UPEC leading to UTI. PMID:29445256

Unsealed tubewells lead to increased fecal contamination of drinking water.

PubMed

Knappett, Peter S K; McKay, Larry D; Layton, Alice; Williams, Daniel E; Alam, Md J; Mailloux, Brian J; Ferguson, Andrew S; Culligan, Patricia J; Serre, Marc L; Emch, Michael; Ahmed, Kazi M; Sayler, Gary S; van Geen, Alexander

2012-12-01

Bangladesh is underlain by shallow aquifers in which millions of drinking water wells are emplaced without annular seals. Fecal contamination has been widely detected in private tubewells. To evaluate the impact of well construction on microbial water quality 35 private tubewells (11 with intact cement platforms, 19 without) and 17 monitoring wells (11 with the annulus sealed with cement, six unsealed) were monitored for culturable Escherichia coli over 18 months. Additionally, two 'snapshot' sampling events were performed on a subset of wells during late-dry and early-wet seasons, wherein the fecal indicator bacteria (FIB) E. coli, Bacteroidales and the pathogenicity genes eltA (enterotoxigenic E. coli; ETEC), ipaH (Shigella) and 40/41 hexon (adenovirus) were detected using quantitative polymerase chain reaction (qPCR). No difference in E. coli detection frequency was found between tubewells with and without platforms. Unsealed private wells, however, contained culturable E. coli more frequently and higher concentrations of FIB than sealed monitoring wells (p < 0.05), suggestive of rapid downward flow along unsealed annuli. As a group the pathogens ETEC, Shigella and adenovirus were detected more frequently (10/22) during the wet season than the dry season (2/20). This suggests proper sealing of private tubewell annuli may lead to substantial improvements in microbial drinking water quality.

Impedance biosensor based on interdigitated electrode array for detection of E.coli O157:H7 in food products

NASA Astrophysics Data System (ADS)

Ghosh Dastider, Shibajyoti; Barizuddin, Syed; Dweik, Majed; Almasri, Mahmoud F.

2012-05-01

An impedance biosensor was designed, fabricated and tested for detection of viable Escherichia coli O157:H7 in food samples. This device consists of interdigitated microelectrode array (IDEA) fabricated using thin layer of sputtered gold, embedded under a polydimethylsiloxane (PDMS) microchannel. The array of electrodes is designed to detect viable EColi in different food products. The active surface area of the detection array was modified using goat anti-E.coli polyclonal IgG antibody. Contaminated food samples were tested by infusing the supernatant containing bacteria over the IDEA's, through the microchannel. Antibody-antigen binding on the electrodes results in impedance change. Four serial concentrations of E.coli contaminated food samples (3x102 CFUmL-1 to 3x105 CFUmL-1) were tested. The biosensor successfully detected the E.coli samples, with the lower detection limit being 3x103 CFUmL-1 (up to 3cells/μl). Comparing the test results with an IDEA impedance biosensor without microchannel (published elsewhere) indicates that this biosensor have two order of magnitude times higher sensitivity. The proposed biosensor provides qualitative and quantitative detection, and potentially could be used for detection of other type of bacteria by immobilizing the specific type of antibody.

An engineered non-oxidative glycolysis pathway for acetone production in Escherichia coli.

PubMed

Yang, Xiaoyan; Yuan, Qianqian; Zheng, Yangyang; Ma, Hongwu; Chen, Tao; Zhao, Xueming

2016-08-01

To find new metabolic engineering strategies to improve the yield of acetone in Escherichia coli. Results of flux balance analysis from a modified Escherichia coli genome-scale metabolic network suggested that the introduction of a non-oxidative glycolysis (NOG) pathway would improve the theoretical acetone yield from 1 to 1.5 mol acetone/mol glucose. By inserting the fxpk gene encoding phosphoketolase from Bifidobacterium adolescentis into the genome, we constructed a NOG pathway in E.coli. The resulting strain produced 47 mM acetone from glucose under aerobic conditions in shake-flasks. The yield of acetone was improved from 0.38 to 0.47 mol acetone/mol glucose which is a significant over the parent strain. Guided by computational analysis of metabolic networks, we introduced a NOG pathway into E. coli and increased the yield of acetone, which demonstrates the importance of modeling analysis for the novel metabolic engineering strategies.

Multiplex Fluorogenic Real-Time PCR for Detection and Quantification of Escherichia coli O157:H7 in Dairy Wastewater Wetlands

PubMed Central

Ibekwe, A. Mark; Watt, Pamela M.; Grieve, Catherine M.; Sharma, Vijay K.; Lyons, Steven R.

2002-01-01

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (CT) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the CT and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10−5 pg of E. coli O157:H7 DNA ml−1 equivalent to approximately 6.4 × 103 CFU of E. coli O157:H7 ml−1 based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 104 CFU g−1. E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g−1 with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems. PMID:12324331

3D-QSAR and docking studies of flavonoids as potent Escherichia coli inhibitors

PubMed Central

Fang, Yajing; Lu, Yulin; Zang, Xixi; Wu, Ting; Qi, XiaoJuan; Pan, Siyi; Xu, Xiaoyun

2016-01-01

Flavonoids are potential antibacterial agents. However, key substituents and mechanism for their antibacterial activity have not been fully investigated. The quantitative structure-activity relationship (QSAR) and molecular docking of flavonoids relating to potent anti-Escherichia coli agents were investigated. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were developed by using the pIC50 values of flavonoids. The cross-validated coefficient (q2) values for CoMFA (0.743) and for CoMSIA (0.708) were achieved, illustrating high predictive capabilities. Selected descriptors for the CoMFA model were ClogP (logarithm of the octanol/water partition coefficient), steric and electrostatic fields, while, ClogP, electrostatic and hydrogen bond donor fields were used for the CoMSIA model. Molecular docking results confirmed that half of the tested flavonoids inhibited DNA gyrase B (GyrB) by interacting with adenosine-triphosphate (ATP) pocket in a same orientation. Polymethoxyl flavones, flavonoid glycosides, isoflavonoids changed their orientation, resulting in a decrease of inhibitory activity. Moreover, docking results showed that 3-hydroxyl, 5-hydroxyl, 7-hydroxyl and 4-carbonyl groups were found to be crucial active substituents of flavonoids by interacting with key residues of GyrB, which were in agreement with the QSAR study results. These results provide valuable information for structure requirements of flavonoids as antibacterial agents. PMID:27049530

Quantitation of bacteria through adsorption of intracellular biomolecules on carbon paste and screen-printed carbon electrodes and voltammetry of redox-active probes.

PubMed

Obuchowska, Agnes

2008-03-01

A new electrochemical method for the quantitation of bacteria that is rapid, inexpensive, and amenable to miniaturization is reported. Cyclic voltammetry was used to quantitate M. luteus, C. sporogenes, and E. coli JM105 in exponential and stationary phases, following exposure of screen-printed carbon working electrodes (SPCEs) to lysed culture samples. Ferricyanide was used as a probe. The detection limits (3s) were calculated and the dynamic ranges for E. coli (exponential and stationary phases), M. luteus (exponential and stationary phases), and C. sporogenes (exponential phase) lysed by lysozyme were 3 x 10(4) to 5 x 10(6) colony-forming units (CFU) mL(-1), 5 x 10(6) to 2 x 10(8) CFU mL(-1) and 3 x 10(3) to 3 x 10(5) CFU mL(-1), respectively. Good overlap was obtained between the calibration curves when the electrochemical signal was plotted against the dry bacterial weight, or between the protein concentration in the bacterial lysate. In contrast, unlysed bacteria did not change the electrochemical signal of ferricyanide. The results indicate that the reduction of the electrochemical signal in the presence of the lysate is mainly due to the fouling of the electrode by proteins. Similar results were obtained with carbon-paste electrodes although detection limits were better with SPCEs. The method described herein was applied to quantitation of bacteria in a cooling tower water sample.

Prevalence and pathogen load of Campylobacter spp., Salmonella enterica and Escherichia coli O157/O145 serogroup in sheep faeces collected at sale yards and in abattoir effluent in Western Australia.

PubMed

Yang, R; Abraham, S; Gardner, G E; Ryan, U; Jacobson, C

2017-05-01

Develop a multiplex quantitative PCR assay to investigate the prevalence and shedding of Escherichia coli O157/O145, Salmonella spp. and Campylobacter spp. in sheep at sale yards and abattoirs. A qPCR for E. coli O157/O145 was developed, validated and multiplexed with an existing qPCR for Campylobacter and Salmonella enterica. The absolute numbers of E. coli O157/O145, Campylobacter and Salmonella in control samples was determined using droplet digital PCR. These were then used as the controls in the multiplex qPCR on a total of 474 sheep faecal samples collected from two saleyards over a 4-month period (April-July 2014) and 96 effluent samples from an abattoir. The mutiplex qPCR was specific with a sensitivity of 5 organisms/μL faecal DNA extract for Campylobacter, S. enterica and E. coli O157/O145. The overall prevalence of Campylobacter, S. enterica and E. coli O157/O145 in faecal samples was 5.7%, 3.6% and 8.4% and in effluent samples was 18.8%, 6.3% and 5.2%, respectively. The pathogen loads of Campylobacter, S. enterica and E. coli O157/O145 in faecal and effluent samples was also determined via mutiplex qPCR. The overall prevalences of Campylobacter, S. enterica and E. coli O157/O145 were generally low (<6%), but point prevalences ranged considerably in healthy sheep (up to 26% for E. coli O157/O145). Further work to determine risk factors for shedding of bacterial organisms in meat sheep in the pre-slaughter period (on-farm, sale yards and lairage at abattoirs) could further reduce the risk of contamination of meat products. © 2017 Australian Veterinary Association.

I See Your Smart Phone and Raise You Smart Bacteria

Science.gov Websites

understanding how bacteria sense their nearest neighbors (including pathogens), a DTRA CB/JSTO-funded research ;wild type" E. coli was tested via reverse transcription quantitative polymerase chain reactions Director of Research, Dale Ormond, kicks off #MATHCOUNTS #NationalCompetition2018 Countdown Round

EMERGING PATHOGENS OF ANIMAL ORIGIN: UNIQUE CHALLENGES FOR QUANTITATIVE MICROBIAL RISK ASSESSMENT

EPA Science Inventory

Recently severe human epidemics caused by waterborne Cryptosporidium parvum and Escherichia coli Strain (157:H7 of animal origin have attracted much attention in the national news media. Among the 130 diseases that can be transmitted to man from domestic and wild animals, a signi...

A critical examination of Escherichia coli esterase activity.

PubMed

Antonczak, Alicja K; Simova, Zuzana; Tippmann, Eric M

2009-10-16

The ability of Escherichia coli to grow on a series of acetylated and glycosylated compounds has been investigated. It is surmised that E. coli maintains low levels of nonspecific esterase activity. This observation may have ramifications for previous reports that relied on nonspecific esterases from E. coli to genetically encode nonnatural amino acids. It had been reported that nonspecific esterases from E. coli deacetylate tri-acetyl O-linked glycosylated serine and threonine in vivo. The glycosylated amino acids were reported to have been genetically encoded into proteins in response to the amber stop codon. However, it is our contention that such amino acids are not utilized in this manner within E. coli. The current results report in vitro analysis of the original enzyme and an in vivo analysis of a glycosylated amino acid. It is concluded that the amber suppression method with nonnatural amino acids may require a caveat for use in certain instances.

A Critical Examination of Escherichia coli Esterase Activity*

PubMed Central

Antonczak, Alicja K.; Simova, Zuzana; Tippmann, Eric M.

2009-01-01

The ability of Escherichia coli to grow on a series of acetylated and glycosylated compounds has been investigated. It is surmised that E. coli maintains low levels of nonspecific esterase activity. This observation may have ramifications for previous reports that relied on nonspecific esterases from E. coli to genetically encode nonnatural amino acids. It had been reported that nonspecific esterases from E. coli deacetylate tri-acetyl O-linked glycosylated serine and threonine in vivo. The glycosylated amino acids were reported to have been genetically encoded into proteins in response to the amber stop codon. However, it is our contention that such amino acids are not utilized in this manner within E. coli. The current results report in vitro analysis of the original enzyme and an in vivo analysis of a glycosylated amino acid. It is concluded that the amber suppression method with nonnatural amino acids may require a caveat for use in certain instances. PMID:19666472

Short-term dynamic behavior of Escherichia coli in response to successive glucose pulses on glucose-limited chemostat cultures.

PubMed

Sunya, Sirichai; Bideaux, Carine; Molina-Jouve, Carole; Gorret, Nathalie

2013-04-15

The effect of repeated glucose perturbations on dynamic behavior of Escherichia coli DPD2085, yciG::LuxCDABE reporter strain, was studied and characterized on a short-time scale using glucose-limited chemostat cultures at dilution rates close to 0.18h(-1). The substrate disturbances were applied on independent steady-state cultures, firstly using a single glucose pulse under different aeration conditions and secondly using repeated glucose pulses under fully aerobic condition. The dynamic responses of E. coli to a single glucose pulse of different intensities (0.25 and 0.6gL(-1)) were significantly similar at macroscopic level, revealing the independency of the macroscopic microbial behavior to the perturbation intensity in the range of tested glucose concentrations. The dynamic responses of E. coli to repeated glucose pulses to simulate fluctuating environments between glucose-limited and glucose-excess conditions were quantified; similar behavior regarding respiration and by-product formations was observed, except for the first perturbation denoted by an overshoot of the specific oxygen uptake rate in the first minutes after the pulse. In addition, transcriptional induction of yciG promoter gene involved in general stress response, σ(S), was monitored through the bioluminescent E. coli strain. This study aims to provide and compare short-term quantitative kinetics data describing the dynamic behavior of E. coli facing repeated transient substrate conditions. Copyright © 2013 Elsevier B.V. All rights reserved.

Bacteriological analysis of indoor and outdoor water parks in Wisconsin.

PubMed

Davis, Tracynda L; Standridge, Jon H; Degnan, Alan J

2009-09-01

Water parks are a rapidly growing element of the United States tourist industry. To reduce incidence of abrasion and impact injuries in such parks, designers are searching for padding materials that can withstand the harsh oxidative environments of chlorinated water. Although padded features help reduce physical injuries, they may also compromise the microbiological safety of water attractions. This study describes bacteriological testing performed on 31 different pad materials, play features and pools from 10 Wisconsin water parks. Materials and surrounding pool waters were sampled and tested quantitatively for total coliforms, Escherichia coli, E. coli 0157:H7, enterococci, staphylococci, heterotrophic bacteria, and Pseudomonas aeruginosa, using standard methods. Each location was sampled during three visits, and results were averaged. Pool waters were within acceptable levels of target organisms and disinfectant residuals, but target organisms were found on water features, even those submerged in chlorinated water. Bacteria were detected more frequently in pools using pad materials compared with pools without. These findings provide data that will help the public health community understand the relations between designs, materials and maintenance of water features. Additionally, the information will help state regulators and owner/operators develop guidelines to improve public health and safety at water parks.

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.

Experimental evidence of the role of pores on movement and distribution of bacteria in soil

NASA Astrophysics Data System (ADS)

Kravchenko, Alexandra N.; Rose, Joan B.; Marsh, Terence L.; Guber, Andrey K.

2014-05-01

It has been generally recognized that micro-scale heterogeneity in soil environments can have a substantial effect on movement, fate, and survival of soil microorganisms. However, only recently the development of tools for micro-scale soil analyses, including X-ray computed micro-tomography (μ-CT), enabled quantitative analyses of these effects. The long-term goal of our work is to explore how differences in micro-scale characteristics of pore structures influence movement, spatial distribution patterns, and activities of soil microorganisms. Using X-ray μ-CT we found that differences in land use and management practices lead to development of contrasting patterns in pore size-distributions within intact soil aggregates. Then our experiments with Escherichia coli added to intact soil aggregates demonstrated that the differences in pore structures can lead to substantial differences in bacteria redistribution and movement within the aggregates. Specifically, we observed more uniform E.coli redistribution in aggregates with homogeneously spread pores, while heterogeneous pore structures resulted in heterogeneous E.coli patterns. Water flow driven by capillary forces through intact aggregate pores appeared to be the main contributor to the movement patterns of the introduced bacteria. Influence of pore structure on E.coli distribution within the aggregates further continued after the aggregates were subjected to saturated water flow. E. coli's resumed movement with saturated water flow and subsequent redistribution within the soil matrix was influenced by porosity, abundance of medium and large pores, pore tortuosity, and flow rates, indicating that greater flow accompanied by less convoluted pores facilitated E. coli transport within the intra-aggregate space. We also found that intra-aggregate heterogeneity of pore structures can have an effect on spatial distribution patterns of indigenous microbial populations. Preliminary analysis showed that in aggregates from an organic agricultural system with cover crops, characterized by greater intra-aggregate pore heterogeneity, bacteria of Actinobacteria and Firmicutes groups were more abundant in presence of large as compared to small pores. In contrast, no differences were observed in the aggregates from conventionally managed soil, overall characterized by homogeneous intra-aggregate pore patterns. Further research efforts are being directed towards quantification of the pore structure effects on activities and community composition of soil microorganisms.

Demand theory of gene regulation. II. Quantitative application to the lactose and maltose operons of Escherichia coli.

PubMed Central

Savageau, M A

1998-01-01

Induction of gene expression can be accomplished either by removing a restraining element (negative mode of control) or by providing a stimulatory element (positive mode of control). According to the demand theory of gene regulation, which was first presented in qualitative form in the 1970s, the negative mode will be selected for the control of a gene whose function is in low demand in the organism's natural environment, whereas the positive mode will be selected for the control of a gene whose function is in high demand. This theory has now been further developed in a quantitative form that reveals the importance of two key parameters: cycle time C, which is the average time for a gene to complete an ON/OFF cycle, and demand D, which is the fraction of the cycle time that the gene is ON. Here we estimate nominal values for the relevant mutation rates and growth rates and apply the quantitative demand theory to the lactose and maltose operons of Escherichia coli. The results define regions of the C vs. D plot within which selection for the wild-type regulatory mechanisms is realizable, and these in turn provide the first estimates for the minimum and maximum values of demand that are required for selection of the positive and negative modes of gene control found in these systems. The ratio of mutation rate to selection coefficient is the most relevant determinant of the realizable region for selection, and the most influential parameter is the selection coefficient that reflects the reduction in growth rate when there is superfluous expression of a gene. The quantitative theory predicts the rate and extent of selection for each mode of control. It also predicts three critical values for the cycle time. The predicted maximum value for the cycle time C is consistent with the lifetime of the host. The predicted minimum value for C is consistent with the time for transit through the intestinal tract without colonization. Finally, the theory predicts an optimum value of C that is in agreement with the observed frequency for E. coli colonizing the human intestinal tract. PMID:9691028

Functional Analysis of Genes Comprising the Locus of Heat Resistance in Escherichia coli.

PubMed

Mercer, Ryan; Nguyen, Oanh; Ou, Qixing; McMullen, Lynn; Gänzle, Michael G

2017-10-15

The locus of heat resistance (LHR) is a 15- to 19-kb genomic island conferring exceptional heat resistance to organisms in the family Enterobacteriaceae , including pathogenic strains of Salmonella enterica and Escherichia coli The complement of LHR-comprising genes that is necessary for heat resistance and the stress-induced or growth-phase-induced expression of LHR-comprising genes are unknown. This study determined the contribution of the seven LHR-comprising genes yfdX1 GI , yfdX2 , hdeD GI , orf11 , trx GI , kefB , and psiE GI by comparing the heat resistances of E. coli strains harboring plasmid-encoded derivatives of the different LHRs in these genes. (Genes carry a subscript "GI" [genomic island] if an ortholog of the same gene is present in genomes of E. coli ) LHR-encoded heat shock proteins sHSP20, ClpK GI , and sHSP GI are not sufficient for the heat resistance phenotype; YfdX1, YfdX2, and HdeD are necessary to complement the LHR heat shock proteins and to impart a high level of resistance. Deletion of trx GI , kefB , and psiE GI from plasmid-encoded copies of the LHR did not significantly affect heat resistance. The effect of the growth phase and the NaCl concentration on expression from the putative LHR promoter p2 was determined by quantitative reverse transcription-PCR and by a plasmid-encoded p2:GFP promoter fusion. The expression levels of exponential- and stationary-phase E. coli cells were not significantly different, but the addition of 1% NaCl significantly increased LHR expression. Remarkably, LHR expression in E. coli was dependent on a chromosomal copy of evgA In conclusion, this study improved our understanding of the genes required for exceptional heat resistance in E. coli and factors that increase their expression in food. IMPORTANCE The locus of heat resistance (LHR) is a genomic island conferring exceptional heat resistance to several foodborne pathogens. The exceptional level of heat resistance provided by the LHR questions the control of pathogens by current food processing and preparation techniques. The function of LHR-comprising genes and their regulation, however, remain largely unknown. This study defines a core complement of LHR-encoded proteins that are necessary for heat resistance and demonstrates that regulation of the LHR in E. coli requires a chromosomal copy of the gene encoding EvgA. This study provides insight into the function of a transmissible genomic island that allows otherwise heat-sensitive enteric bacteria, including pathogens, to lead a thermoduric lifestyle and thus contributes to the detection and control of heat-resistant enteric bacteria in food. Copyright © 2017 American Society for Microbiology.

Functional Analysis of Genes Comprising the Locus of Heat Resistance in Escherichia coli

PubMed Central

Mercer, Ryan; Nguyen, Oanh; Ou, Qixing; McMullen, Lynn

2017-01-01

ABSTRACT The locus of heat resistance (LHR) is a 15- to 19-kb genomic island conferring exceptional heat resistance to organisms in the family Enterobacteriaceae, including pathogenic strains of Salmonella enterica and Escherichia coli. The complement of LHR-comprising genes that is necessary for heat resistance and the stress-induced or growth-phase-induced expression of LHR-comprising genes are unknown. This study determined the contribution of the seven LHR-comprising genes yfdX1GI, yfdX2, hdeDGI, orf11, trxGI, kefB, and psiEGI by comparing the heat resistances of E. coli strains harboring plasmid-encoded derivatives of the different LHRs in these genes. (Genes carry a subscript “GI” [genomic island] if an ortholog of the same gene is present in genomes of E. coli.) LHR-encoded heat shock proteins sHSP20, ClpKGI, and sHSPGI are not sufficient for the heat resistance phenotype; YfdX1, YfdX2, and HdeD are necessary to complement the LHR heat shock proteins and to impart a high level of resistance. Deletion of trxGI, kefB, and psiEGI from plasmid-encoded copies of the LHR did not significantly affect heat resistance. The effect of the growth phase and the NaCl concentration on expression from the putative LHR promoter p2 was determined by quantitative reverse transcription-PCR and by a plasmid-encoded p2:GFP promoter fusion. The expression levels of exponential- and stationary-phase E. coli cells were not significantly different, but the addition of 1% NaCl significantly increased LHR expression. Remarkably, LHR expression in E. coli was dependent on a chromosomal copy of evgA. In conclusion, this study improved our understanding of the genes required for exceptional heat resistance in E. coli and factors that increase their expression in food. IMPORTANCE The locus of heat resistance (LHR) is a genomic island conferring exceptional heat resistance to several foodborne pathogens. The exceptional level of heat resistance provided by the LHR questions the control of pathogens by current food processing and preparation techniques. The function of LHR-comprising genes and their regulation, however, remain largely unknown. This study defines a core complement of LHR-encoded proteins that are necessary for heat resistance and demonstrates that regulation of the LHR in E. coli requires a chromosomal copy of the gene encoding EvgA. This study provides insight into the function of a transmissible genomic island that allows otherwise heat-sensitive enteric bacteria, including pathogens, to lead a thermoduric lifestyle and thus contributes to the detection and control of heat-resistant enteric bacteria in food. PMID:28802266

Total coliform and Escherichia coli contamination in rural well water: analysis for passive surveillance.

PubMed

Invik, Jesse; Barkema, Herman W; Massolo, Alessandro; Neumann, Norman F; Checkley, Sylvia

2017-10-01

With increasing stress on our water resources and recent waterborne disease outbreaks, understanding the epidemiology of waterborne pathogens is crucial to build surveillance systems. The purpose of this study was to explore techniques for describing microbial water quality in rural drinking water wells, based on spatiotemporal analysis, time series analysis and relative risk mapping. Tests results for Escherichia coli and coliforms from private and small public well water samples, collected between 2004 and 2012 in Alberta, Canada, were used for the analysis. Overall, 14.6 and 1.5% of the wells were total coliform and E. coli-positive, respectively. Private well samples were more often total coliform or E. coli-positive compared with untreated public well samples. Using relative risk mapping we were able to identify areas of higher risk for bacterial contamination of groundwater in the province not previously identified. Incorporation of time series analysis demonstrated peak contamination occurring for E. coli in July and a later peak for total coliforms in September, suggesting a temporal dissociation between these indicators in terms of groundwater quality, and highlighting the potential need to increase monitoring during certain periods of the year.

Different cellular origins and functions of extracellular proteins from Escherichia coli O157:H7 and O104:H4 as determined by comparative proteomic analysis

USDA-ARS?s Scientific Manuscript database

Escherichia coli is a diverse species of bacteria, including several pathotypes that cause a variety of diseases in humans. Enterohemorrhagic E. coli (EHEC) and recently emerged shigatoxingenic enteroaggregative E. coli (EAEC) produce Shigatoxins and are major foodborne pathogens that can cause hem...

Escherichia coli B2 strains prevalent in inflammatory bowel disease patients have distinct metabolic capabilities that enable colonization of intestinal mucosa.

PubMed

Fang, Xin; Monk, Jonathan M; Mih, Nathan; Du, Bin; Sastry, Anand V; Kavvas, Erol; Seif, Yara; Smarr, Larry; Palsson, Bernhard O

2018-06-11

Escherichia coli is considered a leading bacterial trigger of inflammatory bowel disease (IBD). E. coli isolates from IBD patients primarily belong to phylogroup B2. Previous studies have focused on broad comparative genomic analysis of E. coli B2 isolates, and identified virulence factors that allow B2 strains to reside within human intestinal mucosa. Metabolic capabilities of E. coli strains have been shown to be related to their colonization site, but remain unexplored in IBD-associated strains. In this study, we utilized pan-genome analysis and genome-scale models (GEMs) of metabolism to study metabolic capabilities of IBD-associated E. coli B2 strains. The study yielded three results: i) Pan-genome analysis of 110 E. coli strains (including 53 isolates from IBD studies) revealed discriminating metabolic genes between B2 strains and other strains; ii) Both comparative genomic analysis and GEMs suggested that B2 strains have an advantage in degrading and utilizing sugars derived from mucus glycan, and iii) GEMs revealed distinct metabolic features in B2 strains that potentially allow them to utilize energy more efficiently. For example, B2 strains lack the enzymes to degrade amadori products, but instead rely on neighboring bacteria to convert these substrates into a more readily usable and potentially less sought after product. Taken together, these results suggest that the metabolic capabilities of B2 strains vary significantly from those of other strains, enabling B2 strains to colonize intestinal mucosa.The results from this study motivate a broad experimental assessment of the nutritional effects on E. coli B2 pathophysiology in IBD patients.

Quantitation of heparosan with heparin lyase III and spectrophotometry.

PubMed

Huang, Haichan; Zhao, Yingying; Lv, Shencong; Zhong, Weihong; Zhang, Fuming; Linhardt, Robert J

2014-02-15

Heparosan is Escherichia coli K5 capsule polysaccharide, which is the key precursor for preparing bioengineered heparin. A rapid and effective quantitative method for detecting heparosan is important in the large-scale production of heparosan. Heparin lyase III (Hep III) effectively catalyzes the heparosan depolymerization, forming unsaturated disaccharides that are measurable using a spectrophotometer at 232 nm. We report a new method for the quantitative detection of heparosan with heparin lyase III and spectrophotometry that is safer and more specific than the traditional carbazole assay. In an optimized detection system, heparosan at a minimum concentration of 0.60 g/L in fermentation broth can be detected. Copyright © 2013 Elsevier Inc. All rights reserved.

Hierarchy of non-glucose sugars in Escherichia coli.

PubMed

Aidelberg, Guy; Towbin, Benjamin D; Rothschild, Daphna; Dekel, Erez; Bren, Anat; Alon, Uri

2014-12-24

Understanding how cells make decisions, and why they make the decisions they make, is of fundamental interest in systems biology. To address this, we study the decisions made by E. coli on which genes to express when presented with two different sugars. It is well-known that glucose, E. coli's preferred carbon source, represses the uptake of other sugars by means of global and gene-specific mechanisms. However, less is known about the utilization of glucose-free sugar mixtures which are found in the natural environment of E. coli and in biotechnology. Here, we combine experiment and theory to map the choices of E. coli among 6 different non-glucose carbon sources. We used robotic assays and fluorescence reporter strains to make precise measurements of promoter activity and growth rate in all pairs of these sugars. We find that the sugars can be ranked in a hierarchy: in a mixture of a higher and a lower sugar, the lower sugar system shows reduced promoter activity. The hierarchy corresponds to the growth rate supported by each sugar- the faster the growth rate, the higher the sugar on the hierarchy. The hierarchy is 'soft' in the sense that the lower sugar promoters are not completely repressed. Measurement of the activity of the master regulator CRP-cAMP shows that the hierarchy can be quantitatively explained based on differential activation of the promoters by CRP-cAMP. Comparing sugar system activation as a function of time in sugar pair mixtures at sub-saturating concentrations, we find cases of sequential activation, and also cases of simultaneous expression of both systems. Such simultaneous expression is not predicted by simple models of growth rate optimization, which predict only sequential activation. We extend these models by suggesting multi-objective optimization for both growing rapidly now and preparing the cell for future growth on the poorer sugar. We find a defined hierarchy of sugar utilization, which can be quantitatively explained by differential activation by the master regulator cAMP-CRP. The present approach can be used to understand cell decisions when presented with mixtures of conditions.

Day-to-Day Dynamics of Commensal Escherichia coli in Zimbabwean Cows Evidence Temporal Fluctuations within a Host-Specific Population Structure.

PubMed

Massot, Méril; Couffignal, Camille; Clermont, Olivier; D'Humières, Camille; Chatel, Jérémie; Plault, Nicolas; Andremont, Antoine; Caron, Alexandre; Mentré, France; Denamur, Erick

2017-07-01

To get insights into the temporal pattern of commensal Escherichia coli populations, we sampled the feces of four healthy cows from the same herd in the Hwange District of Zimbabwe daily over 25 days. The cows had not received antibiotic treatment during the previous 3 months. We performed viable E. coli counts and characterized the 326 isolates originating from the 98 stool samples at a clonal level, screened them for stx and eae genes, and tested them for their antibiotic susceptibilities. We observed that E. coli counts and dominant clones were different among cows, and very few clones were shared. No clone was shared by three or four cows. Clone richness and evenness were not different between cows. Within each host, the variability in the E. coli count was evidenced between days, and no clone was found to be dominant during the entire sampling period, suggesting the existence of clonal interference. Dominant clones tended to persist longer than subdominant ones and were mainly from phylogenetic groups A and B1. Five E. coli clones were found to contain both the stx 1 and stx 2 genes, representing 6.3% of the studied isolates. All cows harbored at least one Shiga toxin-producing E. coli (STEC) strain. Resistance to tetracycline, penicillins, trimethoprim, and sulfonamides was rare and observed in three clones that were shed at low levels in two cows. This study highlights the fact that the commensal E. coli population, including the STEC population, is host specific, is highly dynamic over a short time frame, and rarely carries antibiotic resistance determinants in the absence of antibiotic treatment. IMPORTANCE The literature about the dynamics of commensal Escherichia coli populations is very scarce. Over 25 days, we followed the total E. coli counts daily and characterized the sampled clones in the feces of four cows from the same herd living in the Hwange District of Zimbabwe. This study deals with the day-to-day dynamics of both quantitative and qualitative aspects of E. coli commensal populations, with a focus on both Shiga toxin-producing E. coli and antibiotic-resistant E. coli strains. We show that the structure of these commensal populations was highly specific to the host, even though the cows ate and roamed together, and was highly dynamic between days. Such data are of importance to understand the ecological forces that drive the dynamics of the emergence of E. coli clones of particular interest within the gastrointestinal tract and their transmission between hosts. Copyright © 2017 American Society for Microbiology.

Aptamer immobilization on amino-functionalized metal-organic frameworks: an ultrasensitive platform for the electrochemical diagnostic of Escherichia coli O157:H7.

PubMed

Shahrokhian, Saeed; Ranjbar, Saba

2018-07-07

Herein, we report the development of an electrochemical biosensor for Escherichia coli O157:H7 diagnostic based on amino-functionalized metal-organic frameworks (MOFs) as a new generation of organic-inorganic hybrid nanocomposites. The electrical and morphological properties of MOFs were enhanced by interweaving each isolated MOF crystal with polyaniline (PANI). Subsequent attachment of the amine-modified aptamer to the polyanilinated MOFs was accomplished using glutaraldehyde (GA) as a cross-linking agent. The prepared biocompatible platform was carefully characterized by means of field-emission scanning electron microscopy (FESEM), energy-dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FT-IR), and X-ray powder diffraction (XRD) techniques. The biosensor fabrication and its electrochemical characterizations were monitored by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Differential pulse voltammetry (DPV) was applied to monitoring and quantitation of the interaction between the aptamer and E. coli O157:H7 using methylene blue (MB) as an electrochemical indicator. Changes in the reduction peak current of MB in the presence of E. coli O157:H7 was recorded as an analytical signal and indicated a relationship with the logarithm of the E. coli O157:H7 concentration in the range of 2.1 × 10 1 to 2.1 × 10 7 CFU mL -1 with a LOQ of 21 CFU mL -1 and LOD of 2 CFU mL -1 . The electrochemical aptasensor displayed good recovery values for the detection of E. coli O157:H7 in environmental real samples and also could act as a smart device to investigate the effects of antibacterial agents against E. coli O157:H7.

Released products of pathogenic bacteria stimulate biofilm formation by Escherichia coli K-12 strains.

PubMed

Vacheva, Anna; Ivanova, Radka; Paunova-Krasteva, Tsvetelina; Stoitsova, Stoyanka

2012-06-01

It has recently been shown that pathogens with a limited capacity for sessile growth (like some Escherichia coli O157 strains) can benefit from the presence of other bacteria and form mixed biofilms with companion strains. This study addresses the question whether pathogens may influence attached growth of E. coli non-pathogenic strains via secreted factors. We compared the biofilm-modulating effects of sterile stationary-phase culture media of a biofilm non-producing strain of E. coli O157:H, a laboratory biofilm-producing E. coli K-12 strain and a biofilm-forming strain of the pathogen Yersina enterocolitica O:3. Sessile growth was monitored as biomass (crystal violet assay), exopolysaccharide (ELLA) and morphology (scanning electron and confocal laser microscopy). With two of the E. coli K-12 strains stimulation of biofilm formation by all supernatants was achieved, but only the pathogens' secreted products induced biomass increase in some 'biofilm-deficient' K-12 strains. Lectin-peroxidase labeling indicated changes in colanic acid and poly-N-acetylglucosamine amounts in extracellular matrices. The contribution of indole, protein and polysaccharide to the biofilm-modulating activities of the supernatants was compared. Indole, in concentrations equal to those established in the supernatants, suppressed sessile growth in one K-12 strain. Proteinase K significantly reduced the stimulatory effects of all supernatants, indicating a prominent role of protein/peptide factor(s) in biofilm promotion. The amount of released polysaccharides (rPS) in the supernatants was quantitated then comparable quantities of isolated rPS were applied during biofilm growth. The three rPS had notable strain-specific effects with regard to both the strain-source of the rPS and the E. coli K-12 target strain.

High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli.

PubMed

Harrison, Lucas B; Hanson, Nancy D

2017-06-01

Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification. Copyright © 2017 Harrison and Hanson.

Escherichia coli and fecal-coliform bacteria as indicators of recreational water quality

USGS Publications Warehouse

Francy, D.S.; Myers, Donna N.; Metzker, K.D.

1993-01-01

In 1986, the U.S. Environmental Protection Agency (USEPA) recommended that Escherichia coli (E. coli) be used in place of fecal-coliform bacteria in State recreational water-quality standards as an indicator of fecal contamination. This announcement followed an epidemiological study in which E. coli concentration was shown to be a better predictor of swimming-associated gastrointestinal illness than fecal-coliform concentration. Water-resource managers from Ohio have decided to collect information specific to their waters and decide whether to use E. coli or fecal-coliform bacteria as the basis for State recreational water-quality standards. If one indicator is a better predictor of recreational water quality than the other and if the relation between the two indicators is variable, then the indicator providing the most accurate measure of recreational water quality should be used in water-quality standards. Water-quality studies of the variability of concentrations of E. coli to fecal-coliform bacteria have shown that (1) concentrations of the two indicators are positively correlated, (2) E. coli to fecal-coliform ratios differ considerably from site to site, and (3) the E. coli criteria recommended by USEPA may be more difficult to meet than current (1992) fecal-coliform standards. In this study, a statistical analysis was done on concentrations of E. coli and fecal-coliform bacteria in water samples collected by two government agencies in Ohio-- the U.S. Geological Survey (USGS) and the Ohio River Valley Water Sanitation Commission (ORSANCO). Data were organized initially into five data sets for statistical analysis: (1) Cuyahoga River, (2) Olentangy River, (3) Scioto River, (4) Ohio River at Anderson Ferry, and (5) Ohio River at Cincinnati Water Works and Tanners Creek. The USGS collected the data in sets 1, 2, and 3, whereas ORSANCO collected the data in sets 4 and 5. The relation of E. coli to fecal-coliform concentration was investigated by use of linear-regression analysis and analysis of covariance. Log-transformed E. coli and fecal-coliform concentrations were highly correlated in all data sets (r-values ranged from 0.929 to 0.984). Linear regression analysis on USGS and ORSANCO data sets showed that concentration of E. coli could be predicted from fecal-coliform concentration (coefficients of determination (R2) ranged from 0.863 to 0.970). Results of analysis of covariance (ANCOVA) indicated that the predictive equations among the three USGS data sets and two ORSANCO data sets were not significantly different and that the data could be pooled into two large data sets, one for USGS data and one for ORSANCO data. However, results of ANCOVA indicated that USGS and ORSANCO data could not be pooled into one large data set. Predictions of E. coli concentrations calculated for USGS And ORSANCO regression relations, based on fecal-coliform concentrations set to equal Ohio water-quality standards, further showed the differences in E. coli to fecal-coliform relations among data sets. For USGS data, a predicted geometric mean of 176 col/100 mL (number of colonies per 100 milliliters) was greater than the current geometric-mean E. coli standard for bathing water of 126 col/100mL. In contrast, for ORSANCO data, the predicted geometric mean of 101 col/100 mL was less than the current E. coli standard. The risk of illness associated with predicted E. coli concentrations for USGS and ORSANCO data was evaluated by use of the USEPA regression equation that predicts swimming-related gastroenteritis rates from E. coli concentrations.1 The predicted geometric-mean E. coli concentrations for bathing water of 176 col/100 mL for USGS data and 101 col/100 mL for ORSANCO data would allow 9.4 and 7.1 gastrointestinal illnesses per 1,000 swimmers, respectively. This prediction compares well with the illness rate of 8 individuals per 1,000 swimmers estimated by the USEPA for an E. coli concentration of 126 col/100 mL. Therefore, the

Evaluation of diarrheagenic E. coli in riversheds by quantitative PCR in combination with enrichment.

PubMed

Hsu, Shih-Yung; Hsu, Bing-Mu; Ji, Wen-Tsai; Hsu, Tsui-Kang; Kao, Po-Min; Shen, Tzung-Yu; Fan, Cheng-Wei; Huang, Yu-Li

2014-01-01

Diarrheagenic Escherichia coli (DEC) is a group of the most common agents of diarrhea. Highly virulent DEC strains could cause illness with dozens of organisms. Waterborne DEC may be detected using polymerase chain reaction (PCR); however, environmental contaminants can interfere with PCR reaction, thus causing the prevalence of DEC to be underestimated. In this study, we propose an approach to efficiently quantify trace amounts of DEC. An enrichment procedure was performed to amplify total E. coli including DEC in the water samples. By normalizing the number of pathotype-specific genes to the amplification rate of a housekeeping gene in all E. coli, the quantity of DEC in original samples could be assessed. This method allows detection of trace amounts of DEC in receiving waters. The results showed that the presence of DEC in water samples was partially associated with riverside settlement. The DEC concentration was substantially higher at a few sampling sites, suggesting that evaluation of DEC along the river may help identify previously unknown pollution sources. Although the sustainability of DEC in the receiving waters may be low, the risk of DEC infection from the watershed warrants further examination.

Quantitative risk assessment for Escherichia coli O157:H7 in frozen ground beef patties consumed by young children in French households.

PubMed

Delignette-Muller, M L; Cornu, M

2008-11-30

A quantitative risk assessment for Escherichia coli O157:H7 in frozen ground beef patties consumed by children under 10 years of age in French households was conducted by a national study group describing an outbreak which occurred in France in 2005. Our exposure assessment model incorporates results from French surveys on consumption frequency of ground beef patties, serving size and consumption preference, microbial destruction experiments and microbial counts on patties sampled from the industrial batch which were responsible for the outbreak. Two different exposure models were proposed, respectively for children under the age of 5 and for children between 5 and 10 years. For each of these two age groups, a single-hit dose-response model was proposed to describe the probability of hemolytic and uremic syndrome (HUS) as a function of the ingested dose. For each group, the single parameter of this model was estimated by Bayesian inference, using the results of the exposure assessment and the epidemiological data collected during the outbreak. Results show that children under 5 years of age are roughly 5 times more susceptible to the pathogen than children over 5 years. Exposure and dose-response models were used in a scenario analysis in order to validate the use of the model and to propose appropriate guidelines in order to prevent new outbreaks. The impact of the cooking preference was evaluated, showing that only a well-done cooking notably reduces the HUS risk, without annulling it. For each age group, a relation between the mean individual HUS risk per serving and the contamination level in a ground beef batch was proposed, as a tool to help French risk managers.

Non-stochastic sampling error in quantal analyses for Campylobacter species on poultry products

USDA-ARS?s Scientific Manuscript database

Using primers and fluorescent probes specific for the most common foodborne Campylobacter species (C. jejuni = Cj and C. coli = Cc), we developed a multiplex, most probable number (MPN) assay using quantitative PCR (qPCR) as the determinant for binomial detection: number of p positives out of n = 6 ...

Enteroaggregative Escherichia coli (EAEC) impairs growth while malnutrition worsens EAEC infection: a novel murine model of the infection malnutrition cycle.

PubMed

Roche, James K; Cabel, Ace; Sevilleja, Jesus; Nataro, James; Guerrant, Richard L

2010-08-15

Enteroaggregative Escherichia coli is emerging as an increasingly recognized cause of persistent, mildly inflammatory diarrhea in the United States, especially among patients with AIDS, as well as among children, for whom it is accompanied by growth shortfalls. We describe a novel model of disease induced in neonatal and weaned C57BL/6 mice by pathogenic strains of enteroaggregative E. coli 042 and JM221. Enteroaggregative E. coli caused growth impairment (up to 47%), persistent stool shedding (for >3 weeks), and a substantial tissue burden of organisms (150,000 organisms per milligram of tissue), as well as histopathological changes in the colonic epithelium (days 4 and 6 of infection) using the model. Undernutrition in neonatal mice, as well as in weaned mice, intensified infection by 1-4 logs, as assessed by quantitative polymerase chain reaction for fecal shedding of organisms. Growth impairment was dependent on both microorganism burden and challenge dose. Both neonatal and weaned mice provide models for a vicious cycle of enteroaggregative E. coli infection that causes growth shortfalls and undernutrition, thus worsening infection. Hence, these neonatal and weaned mice provide the opportunity to dissect mechanisms of this cycle in childhood malnutrition, as well as to define the role played by innate and acquired host defenses in this important infection.

Stock culture heterogeneity rather than new mutational variation complicates short-term cell physiology studies of Escherichia coli K-12 MG1655 in continuous culture.

PubMed

Nahku, Ranno; Peebo, Karl; Valgepea, Kaspar; Barrick, Jeffrey E; Adamberg, Kaarel; Vilu, Raivo

2011-09-01

Nutrient-limited continuous cultures in chemostats have been used to study microbial cell physiology for over 60 years. Genome instability and genetic heterogeneity are possible uncontrolled factors in continuous cultivation experiments. We investigated these issues by using high-throughput (HT) DNA sequencing to characterize samples from different phases of a glucose-limited accelerostat (A-stat) experiment with Escherichia coli K-12 MG1655 and a duration regularly used in cell physiology studies (20 generations of continuous cultivation). Seven consensus mutations from the reference sequence and five subpopulations characterized by different mutations were detected in the HT-sequenced samples. This genetic heterogeneity was confirmed to result from the stock culture by Sanger sequencing. All the subpopulations in which allele frequencies increased (betA, cspG/cspH, glyA) during the experiment were also present at the end of replicate A-stats, indicating that no new subpopulations emerged during our experiments. The fact that ~31 % of the cells in our initial cultures obtained directly from a culture stock centre were mutants raises concerns that even if cultivations are started from single colonies, there is a significant chance of picking a mutant clone with an altered phenotype. Our results show that current HT DNA sequencing technology allows accurate subpopulation analysis and demonstrates that a glucose-limited E. coli K-12 MG1655 A-stat experiment with a duration of tens of generations is suitable for studying cell physiology and collecting quantitative data for metabolic modelling without interference from new mutations.

Stock culture heterogeneity rather than new mutational variation complicates short-term cell physiology studies of Escherichia coli K-12 MG1655 in continuous culture

PubMed Central

Nahku, Ranno; Peebo, Karl; Valgepea, Kaspar; Barrick, Jeffrey E.; Adamberg, Kaarel

2011-01-01

Nutrient-limited continuous cultures in chemostats have been used to study microbial cell physiology for over 60 years. Genome instability and genetic heterogeneity are possible uncontrolled factors in continuous cultivation experiments. We investigated these issues by using high-throughput (HT) DNA sequencing to characterize samples from different phases of a glucose-limited accelerostat (A-stat) experiment with Escherichia coli K-12 MG1655 and a duration regularly used in cell physiology studies (20 generations of continuous cultivation). Seven consensus mutations from the reference sequence and five subpopulations characterized by different mutations were detected in the HT-sequenced samples. This genetic heterogeneity was confirmed to result from the stock culture by Sanger sequencing. All the subpopulations in which allele frequencies increased (betA, cspG/cspH, glyA) during the experiment were also present at the end of replicate A-stats, indicating that no new subpopulations emerged during our experiments. The fact that ~31 % of the cells in our initial cultures obtained directly from a culture stock centre were mutants raises concerns that even if cultivations are started from single colonies, there is a significant chance of picking a mutant clone with an altered phenotype. Our results show that current HT DNA sequencing technology allows accurate subpopulation analysis and demonstrates that a glucose-limited E. coli K-12 MG1655 A-stat experiment with a duration of tens of generations is suitable for studying cell physiology and collecting quantitative data for metabolic modelling without interference from new mutations. PMID:21700661

One-step production of immobilized alpha-amylase in recombinant Escherichia coli.

PubMed

Rasiah, Indira A; Rehm, Bernd H A

2009-04-01

Industrial enzymes are often immobilized via chemical cross-linking onto solid supports to enhance stability and facilitate repeated use in bioreactors. For starch-degrading enzymes, immobilization usually places constraints on enzymatic conversion due to the limited diffusion of the macromolecular substrate through available supports. This study describes the one-step immobilization of a highly thermostable alpha-amylase (BLA) from Bacillus licheniformis and its functional display on the surface of polyester beads inside engineered Escherichia coli. An optimized BLA variant (Termamyl) was N-terminally fused to the polyester granule-forming enzyme PhaC of Cupriavidus necator. The fusion protein lacking the signal sequence mediated formation of stable polyester beads exhibiting alpha-amylase activity. The alpha-amylase beads were assessed with respect to alpha-amylase activity, which was demonstrated qualitatively and quantitatively. The immobilized alpha-amylase showed Michaelis-Menten enzyme kinetics exerting a V(max) of about 506 mU/mg of bead protein with a K(m) of about 5 microM, consistent with that of free alpha-amylase. The stability of the enzyme at 85 degrees C and the capacity for repeated usage in a starch liquefaction process were also demonstrated. In addition, structural integrity and functionality of the beads at extremes of pH and temperature, demonstrating their suitability for industrial use, were confirmed by electron microscopy and protein/enzyme analysis. This study proposes a novel, cost-effective method for the production of immobilized alpha-amylase in a single step by using the polyester granules forming protein PhaC as a fusion partner in engineered E. coli.

Characterizing spatial structure of sediment E. coli populations to inform sampling design.

PubMed

Piorkowski, Gregory S; Jamieson, Rob C; Hansen, Lisbeth Truelstrup; Bezanson, Greg S; Yost, Chris K

2014-01-01

Escherichia coli can persist in streambed sediments and influence water quality monitoring programs through their resuspension into overlying waters. This study examined the spatial patterns in E. coli concentration and population structure within streambed morphological features during baseflow and following stormflow to inform sampling strategies for representative characterization of E. coli populations within a stream reach. E. coli concentrations in bed sediments were significantly different (p = 0.002) among monitoring sites during baseflow, and significant interactive effects (p = 0.002) occurred among monitoring sites and morphological features following stormflow. Least absolute shrinkage and selection operator (LASSO) regression revealed that water velocity and effective particle size (D 10) explained E. coli concentration during baseflow, whereas sediment organic carbon, water velocity and median particle diameter (D 50) were important explanatory variables following stormflow. Principle Coordinate Analysis illustrated the site-scale differences in sediment E. coli populations between disconnected stream segments. Also, E. coli populations were similar among depositional features within a reach, but differed in relation to high velocity features (e.g., riffles). Canonical correspondence analysis resolved that E. coli population structure was primarily explained by spatial (26.9–31.7 %) over environmental variables (9.2–13.1 %). Spatial autocorrelation existed among monitoring sites and morphological features for both sampling events, and gradients in mean particle diameter and water velocity influenced E. coli population structure for the baseflow and stormflow sampling events, respectively. Representative characterization of streambed E. coli requires sampling of depositional and high velocity environments to accommodate strain selectivity among these features owing to sediment and water velocity heterogeneity.

The Glyphosate-Based Herbicide Roundup Does not Elevate Genome-Wide Mutagenesis of Escherichia coli.

PubMed

Tincher, Clayton; Long, Hongan; Behringer, Megan; Walker, Noah; Lynch, Michael

2017-10-05

Mutations induced by pollutants may promote pathogen evolution, for example by accelerating mutations conferring antibiotic resistance. Generally, evaluating the genome-wide mutagenic effects of long-term sublethal pollutant exposure at single-nucleotide resolution is extremely difficult. To overcome this technical barrier, we use the mutation accumulation/whole-genome sequencing (MA/WGS) method as a mutagenicity test, to quantitatively evaluate genome-wide mutagenesis of Escherichia coli after long-term exposure to a wide gradient of the glyphosate-based herbicide (GBH) Roundup Concentrate Plus. The genome-wide mutation rate decreases as GBH concentration increases, suggesting that even long-term GBH exposure does not compromise the genome stability of bacteria. Copyright © 2017 Tincher et al.

The Evolutionary Divergence of Shiga Toxin-Producing Escherichia coli Is Reflected in Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Spacer Composition

PubMed Central

Yin, Shuang; Jensen, Mark A.; Bai, Jiawei; DebRoy, Chitrita; Barrangou, Rodolphe

2013-01-01

The Shiga toxin-producing Escherichia coli (STEC) strains, including those of O157:H7 and the “big six” serogroups (i.e., serogroups O26, O45, O103, O111, O121, and O145), are a group of pathogens designated food adulterants in the United States. The relatively conserved nature of clustered regularly interspaced short palindromic repeats (CRISPRs) in phylogenetically related E. coli strains makes them potential subtyping markers for STEC detection, and a quantitative PCR (qPCR)-based assay was previously developed for O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 isolates. To better evaluate the sensitivity and specificity of this qPCR method, the CRISPR loci of 252 O157 and big-six STEC isolates were sequenced and analyzed along with 563 CRISPR1 and 624 CRISPR2 sequences available in GenBank. General conservation of spacer content and order was observed within each O157 and big-six serogroup, validating the qPCR method. Meanwhile, it was found that spacer deletion, the presence of an insertion sequence, and distinct alleles within a serogroup are sources of false-negative reactions. Conservation of CRISPR arrays among isolates expressing the same flagellar antigen, specifically, H7, H2, and H11, suggested that these isolates share an ancestor and provided an explanation for the false positives previously observed in the qPCR results. An analysis of spacer distribution across E. coli strains provided limited evidence for temporal spacer acquisition. Conversely, comparison of CRISPR sequences between strains along the stepwise evolution of O157:H7 from its O55:H7 ancestor revealed that, over this ∼7,000-year span, spacer deletion was the primary force generating CRISPR diversity. PMID:23851088

The evolutionary divergence of Shiga toxin-producing Escherichia coli is reflected in clustered regularly interspaced short palindromic repeat (CRISPR) spacer composition.

PubMed

Yin, Shuang; Jensen, Mark A; Bai, Jiawei; Debroy, Chitrita; Barrangou, Rodolphe; Dudley, Edward G

2013-09-01

The Shiga toxin-producing Escherichia coli (STEC) strains, including those of O157:H7 and the "big six" serogroups (i.e., serogroups O26, O45, O103, O111, O121, and O145), are a group of pathogens designated food adulterants in the United States. The relatively conserved nature of clustered regularly interspaced short palindromic repeats (CRISPRs) in phylogenetically related E. coli strains makes them potential subtyping markers for STEC detection, and a quantitative PCR (qPCR)-based assay was previously developed for O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 isolates. To better evaluate the sensitivity and specificity of this qPCR method, the CRISPR loci of 252 O157 and big-six STEC isolates were sequenced and analyzed along with 563 CRISPR1 and 624 CRISPR2 sequences available in GenBank. General conservation of spacer content and order was observed within each O157 and big-six serogroup, validating the qPCR method. Meanwhile, it was found that spacer deletion, the presence of an insertion sequence, and distinct alleles within a serogroup are sources of false-negative reactions. Conservation of CRISPR arrays among isolates expressing the same flagellar antigen, specifically, H7, H2, and H11, suggested that these isolates share an ancestor and provided an explanation for the false positives previously observed in the qPCR results. An analysis of spacer distribution across E. coli strains provided limited evidence for temporal spacer acquisition. Conversely, comparison of CRISPR sequences between strains along the stepwise evolution of O157:H7 from its O55:H7 ancestor revealed that, over this ∼7,000-year span, spacer deletion was the primary force generating CRISPR diversity.

Comparison and Recovery of Escherichia coli and Thermotolerant Coliforms in Water with a Chromogenic Medium Incubated at 41 and 44.5°C

PubMed Central

Alonso, Jose L.; Soriano, Adela; Carbajo, Oscar; Amoros, Inmaculada; Garelick, Hemda

1999-01-01

This study compared the performance of a commercial chromogenic medium, CHROMagarECC (CECC), and CECC supplemented with sodium pyruvate (CECCP) with the membrane filtration lauryl sulfate-based medium (mLSA) for enumeration of Escherichia coli and non-E. coli thermotolerant coliforms (KEC). To establish that we could recover the maximum KEC and E. coli population, we compared two incubation temperature regimens, 41 and 44.5°C. Statistical analysis by the Fisher test of data did not demonstrate any statistically significant differences (P = 0.05) in the enumeration of E. coli for the different media (CECC and CECCP) and incubation temperatures. Variance analysis of data performed on KEC counts showed significant differences (P = 0.01) between KEC counts at 41 and 44.5°C on both CECC and CECCP. Analysis of variance demonstrated statistically significant differences (P = 0.05) in the enumeration of total thermotolerant coliforms (TTCs) on CECC and CECCP compared with mLSA. Target colonies were confirmed to be E. coli at a rate of 91.5% and KEC of likely fecal origin at a rate of 77.4% when using CECCP incubated at 41°C. The results of this study showed that CECCP agar incubated at 41°C is efficient for the simultaneous enumeration of E. coli and KEC from river and marine waters. PMID:10427079

Global dynamics of the Escherichia coli proteome and phosphoproteome during growth in minimal medium.

PubMed

Soares, Nelson C; Spät, Philipp; Krug, Karsten; Macek, Boris

2013-06-07

Recent phosphoproteomics studies have generated relatively large data sets of bacterial proteins phosphorylated on serine, threonine, and tyrosine, implicating this type of phosphorylation in the regulation of vital processes of a bacterial cell; however, most phosphoproteomics studies in bacteria were so far qualitative. Here we applied stable isotope labeling by amino acids in cell culture (SILAC) to perform a quantitative analysis of proteome and phosphoproteome dynamics of Escherichia coli during five distinct phases of growth in the minimal medium. Combining two triple-SILAC experiments, we detected a total of 2118 proteins and quantified relative dynamics of 1984 proteins in all measured phases of growth, including 570 proteins associated with cell wall and membrane. In the phosphoproteomic experiment, we detected 150 Ser/Thr/Tyr phosphorylation events, of which 108 were localized to a specific amino acid residue and 76 were quantified in all phases of growth. Clustering analysis of SILAC ratios revealed distinct sets of coregulated proteins for each analyzed phase of growth and overrepresentation of membrane proteins in transition between exponential and stationary phases. The proteomics data indicated that proteins related to stress response typically associated with the stationary phase, including RpoS-dependent proteins, had increasing levels already during earlier phases of growth. Application of SILAC enabled us to measure median occupancies of phosphorylation sites, which were generally low (<12%). Interestingly, the phosphoproteome analysis showed a global increase of protein phosphorylation levels in the late stationary phase, pointing to a likely role of this modification in later phases of growth.

40 CFR 141.706 - Reporting source water monitoring results.

Code of Federal Regulations, 2010 CFR

2010-07-01

... separation. (2) Systems must report the following data elements for each E. coli analysis: Data element. 1.... Source type (flowing stream, lake/reservoir, GWUDI). 7. E. coli/100 mL. 8. Turbidity. 1 1 Systems serving... to report turbidity with their E. coli results. ...

40 CFR 141.706 - Reporting source water monitoring results.

Code of Federal Regulations, 2011 CFR

2011-07-01

... separation. (2) Systems must report the following data elements for each E. coli analysis: Data element. 1.... Source type (flowing stream, lake/reservoir, GWUDI). 7. E. coli/100 mL. 8. Turbidity. 1 1 Systems serving... to report turbidity with their E. coli results. ...

Comparison and Cost Analysis of Drinking Water Quality Monitoring Requirements versus Practice in Seven Developing Countries

PubMed Central

Crocker, Jonny; Bartram, Jamie

2014-01-01

Drinking water quality monitoring programs aim to support provision of safe drinking water by informing water quality management. Little evidence or guidance exists on best monitoring practices for low resource settings. Lack of financial, human, and technological resources reduce a country’s ability to monitor water supply. Monitoring activities were characterized in Cambodia, Colombia, India (three states), Jordan, Peru, South Africa, and Uganda according to water sector responsibilities, monitoring approaches, and marginal cost. The seven study countries were selected to represent a range of low resource settings. The focus was on monitoring of microbiological parameters, such as E. coli, coliforms, and H2S-producing microorganisms. Data collection involved qualitative and quantitative methods. Across seven study countries, few distinct approaches to monitoring were observed, and in all but one country all monitoring relied on fixed laboratories for sample analysis. Compliance with monitoring requirements was highest for operational monitoring of large water supplies in urban areas. Sample transport and labor for sample collection and analysis together constitute approximately 75% of marginal costs, which exclude capital costs. There is potential for substantive optimization of monitoring programs by considering field-based testing and by fundamentally reconsidering monitoring approaches for non-piped supplies. This is the first study to look quantitatively at water quality monitoring practices in multiple developing countries. PMID:25046632

Temporal variations of Escherichia coli concentrations in a large Midwestern river

NASA Astrophysics Data System (ADS)

Schilling, Keith E.; Zhang, You-Kuan; Hill, Dennis R.; Jones, Christopher S.; Wolter, Calvin F.

2009-02-01

SummaryThe Raccoon River used by the Des Moines Water Works to serve more than 400,000 people in central Iowa is threatened by contamination from Escherichia coli bacteria from point and nonpoint sources. The 9389 km 2 watershed is highly agricultural, with 73% of the land in row crop production and widespread animal production. Results from 2155 grab samples from 1997 to 2005 for E. coli analysis were examined for temporal variations. E. coli concentrations were found to vary across years, seasons, and flow conditions, with a 9-year mean value of 1156 most probable number (MPN)/100 ml. Monthly concentrations exhibited clear seasonality with highest values in May through July. Although E. coli concentrations were higher during periods of greater discharge, the relation of log E. coli to log discharge was not particularly strong ( r2 = 0.35). The variogram of E. coli concentrations showed temporal correlation within a span of 4 days suggesting that concentrations measured on 1 day may be related in time to concentrations measured up to 3 days later and beyond 4 days the concentrations vary randomly. The spectral analysis of the time series of E. coli was also carried out and was fitted well with the spectrum of an exponential covariance function. Deciphering temporal patterns and correlation of E. coli bacteria in streams may be useful for developing future monitoring strategies to track concentration patterns and loads.

Quantitative Assessment of Thermodynamic Constraints on the Solution Space of Genome-Scale Metabolic Models

PubMed Central

Hamilton, Joshua J.; Dwivedi, Vivek; Reed, Jennifer L.

2013-01-01

Constraint-based methods provide powerful computational techniques to allow understanding and prediction of cellular behavior. These methods rely on physiochemical constraints to eliminate infeasible behaviors from the space of available behaviors. One such constraint is thermodynamic feasibility, the requirement that intracellular flux distributions obey the laws of thermodynamics. The past decade has seen several constraint-based methods that interpret this constraint in different ways, including those that are limited to small networks, rely on predefined reaction directions, and/or neglect the relationship between reaction free energies and metabolite concentrations. In this work, we utilize one such approach, thermodynamics-based metabolic flux analysis (TMFA), to make genome-scale, quantitative predictions about metabolite concentrations and reaction free energies in the absence of prior knowledge of reaction directions, while accounting for uncertainties in thermodynamic estimates. We applied TMFA to a genome-scale network reconstruction of Escherichia coli and examined the effect of thermodynamic constraints on the flux space. We also assessed the predictive performance of TMFA against gene essentiality and quantitative metabolomics data, under both aerobic and anaerobic, and optimal and suboptimal growth conditions. Based on these results, we propose that TMFA is a useful tool for validating phenotypes and generating hypotheses, and that additional types of data and constraints can improve predictions of metabolite concentrations. PMID:23870272

Occurrence and molecular analysis of Balantidium coli in mountain gorilla (Gorilla beringei beringei) in the Volcanoes National Park, Rwanda.

PubMed

Hassell, James M; Blake, Damer P; Cranfield, Michael R; Ramer, Jan; Hogan, Jennifer N; Noheli, Jean Bosco; Waters, Michael; Hermosilla, Carlos

2013-10-01

Cysts morphologically resembling Balantidium coli were identified in the feces of a mountain gorilla (Gorilla beringei beringei). Confirmatory PCR and sequencing revealed two distinct B. coli-specific sequences (ITS-1, sub-types A0 and B1). This represents the first report of B. coli in this species, raising the possibility of infection from a reservoir host.

Characterization of Escherichia coli isolates from different fecal sources by means of classification tree analysis of fatty acid methyl ester (FAME) profiles.

PubMed

Seurinck, Sylvie; Deschepper, Ellen; Deboch, Bishaw; Verstraete, Willy; Siciliano, Steven

2006-03-01

Microbial source tracking (MST) methods need to be rapid, inexpensive and accurate. Unfortunately, many MST methods provide a wealth of information that is difficult to interpret by the regulators who use this information to make decisions. This paper describes the use of classification tree analysis to interpret the results of a MST method based on fatty acid methyl ester (FAME) profiles of Escherichia coli isolates, and to present results in a format readily interpretable by water quality managers. Raw sewage E. coli isolates and animal E. coli isolates from cow, dog, gull, and horse were isolated and their FAME profiles collected. Correct classification rates determined with leaveone-out cross-validation resulted in an overall low correct classification rate of 61%. A higher overall correct classification rate of 85% was obtained when the animal isolates were pooled together and compared to the raw sewage isolates. Bootstrap aggregation or adaptive resampling and combining of the FAME profile data increased correct classification rates substantially. Other MST methods may be better suited to differentiate between different fecal sources but classification tree analysis has enabled us to distinguish raw sewage from animal E. coli isolates, which previously had not been possible with other multivariate methods such as principal component analysis and cluster analysis.

[Presence of fecal coliforms, Escherichia coli and DNAse- and coagulase-positive Staphylococcus aureus, in "colonial" cheese sold in the city of Blumenau, Estado de Santa Catarina, Brazil].

PubMed

Reibnitz, M G; Tavares, L B; García, J A

1998-01-01

Twenty cheese samples were collected at Blumenau (SC) and were submitted to analysis in order to verify the presence of fecal coliforms, Escherichia coli and Staphylococcus aureus. Among the 20 samples of cheese, analysis revealed that 70% and 20% respectively, were not within present legal specifications (Norma 001/87-DNVSA) for fecal coliforms and Escherichia coli. For Staphylococcus, 95% of the samples were not within present legal specifications.

Fecal indicators and zoonotic pathogens in household drinking water taps fed from rainwater tanks in Southeast Queensland, Australia.

PubMed

Ahmed, W; Hodgers, L; Sidhu, J P S; Toze, S

2012-01-01

In this study, the microbiological quality of household tap water samples fed from rainwater tanks was assessed by monitoring the numbers of Escherichia coli bacteria and enterococci from 24 households in Southeast Queensland (SEQ), Australia. Quantitative PCR (qPCR) was also used for the quantitative detection of zoonotic pathogens in water samples from rainwater tanks and connected household taps. The numbers of zoonotic pathogens were also estimated in fecal samples from possums and various species of birds by using qPCR, as possums and birds are considered to be the potential sources of fecal contamination in roof-harvested rainwater (RHRW). Among the 24 households, 63% of rainwater tank and 58% of connected household tap water (CHTW) samples contained E. coli and exceeded Australian drinking water guidelines of <1 CFU E. coli per 100 ml water. Similarly, 92% of rainwater tanks and 83% of CHTW samples also contained enterococci. In all, 21%, 4%, and 13% of rainwater tank samples contained Campylobacter spp., Salmonella spp., and Giardia lamblia, respectively. Similarly, 21% of rainwater tank and 13% of CHTW samples contained Campylobacter spp. and G. lamblia, respectively. The number of E. coli (P = 0.78), Enterococcus (P = 0.64), Campylobacter (P = 0.44), and G. lamblia (P = 0.50) cells in rainwater tanks did not differ significantly from the numbers observed in the CHTW samples. Among the 40 possum fecal samples tested, Campylobacter spp., Cryptosporidium parvum, and G. lamblia were detected in 60%, 13%, and 30% of samples, respectively. Among the 38 bird fecal samples tested, Campylobacter spp., Salmonella spp., C. parvum, and G. lamblia were detected in 24%, 11%, 5%, and 13% of the samples, respectively. Household tap water samples fed from rainwater tanks tested in the study appeared to be highly variable. Regular cleaning of roofs and gutters, along with pruning of overhanging tree branches, might also prove effective in reducing animal fecal contamination of rainwater tanks.

Fecal Indicators and Zoonotic Pathogens in Household Drinking Water Taps Fed from Rainwater Tanks in Southeast Queensland, Australia

PubMed Central

Hodgers, L.; Sidhu, J. P. S.; Toze, S.

2012-01-01

In this study, the microbiological quality of household tap water samples fed from rainwater tanks was assessed by monitoring the numbers of Escherichia coli bacteria and enterococci from 24 households in Southeast Queensland (SEQ), Australia. Quantitative PCR (qPCR) was also used for the quantitative detection of zoonotic pathogens in water samples from rainwater tanks and connected household taps. The numbers of zoonotic pathogens were also estimated in fecal samples from possums and various species of birds by using qPCR, as possums and birds are considered to be the potential sources of fecal contamination in roof-harvested rainwater (RHRW). Among the 24 households, 63% of rainwater tank and 58% of connected household tap water (CHTW) samples contained E. coli and exceeded Australian drinking water guidelines of <1 CFU E. coli per 100 ml water. Similarly, 92% of rainwater tanks and 83% of CHTW samples also contained enterococci. In all, 21%, 4%, and 13% of rainwater tank samples contained Campylobacter spp., Salmonella spp., and Giardia lamblia, respectively. Similarly, 21% of rainwater tank and 13% of CHTW samples contained Campylobacter spp. and G. lamblia, respectively. The number of E. coli (P = 0.78), Enterococcus (P = 0.64), Campylobacter (P = 0.44), and G. lamblia (P = 0.50) cells in rainwater tanks did not differ significantly from the numbers observed in the CHTW samples. Among the 40 possum fecal samples tested, Campylobacter spp., Cryptosporidium parvum, and G. lamblia were detected in 60%, 13%, and 30% of samples, respectively. Among the 38 bird fecal samples tested, Campylobacter spp., Salmonella spp., C. parvum, and G. lamblia were detected in 24%, 11%, 5%, and 13% of the samples, respectively. Household tap water samples fed from rainwater tanks tested in the study appeared to be highly variable. Regular cleaning of roofs and gutters, along with pruning of overhanging tree branches, might also prove effective in reducing animal fecal contamination of rainwater tanks. PMID:22020514

Influence of the amyloid dye Congo red on curli, cellulose, and the extracellular matrix in E. coli during growth and matrix purification.

PubMed

Reichhardt, Courtney; McCrate, Oscar A; Zhou, Xiaoxue; Lee, Jessica; Thongsomboon, Wiriya; Cegelski, Lynette

2016-11-01

Microbial biofilms are communities of cells characterized by a hallmark extracellular matrix (ECM) that confers functional attributes to the community, including enhanced cohesion, adherence to surfaces, and resistance to external stresses. Understanding the composition and properties of the biofilm ECM is crucial to understanding how it functions and protects cells. New methods to isolate and characterize ECM are emerging for different biofilm systems. Solid-state nuclear magnetic resonance was used to quantitatively track the isolation of the insoluble ECM from the uropathogenic Escherichia coli strain UTI89 and understand the role of Congo red in purification protocols. UTI89 assembles amyloid-integrated biofilms when grown on YESCA nutrient agar. The ECM contains curli amyloid fibers and a modified form of cellulose. Biofilms formed by UTI89 and other E. coli and Salmonella strains are often grown in the presence of Congo red to visually emphasize wrinkled agar morphologies and to score the production of ECM. Congo red is a hallmark amyloid-binding dye and binds to curli, yet also binds to cellulose. We found that growth in Congo red enabled more facile extraction of the ECM from UTI89 biofilms and facilitates isolation of cellulose from the curli mutant, UTI89ΔcsgA. Yet, Congo red has no influence on the isolation of curli from curli-producing cells that do not produce cellulose. Sodium dodecyl sulfate can remove Congo red from curli, but not from cellulose. Thus, Congo red binds strongly to cellulose and possibly weakens cellulose interactions with the cell surface, enabling more complete removal of the ECM. The use of Congo red as an extracellular matrix purification aid may be applied broadly to other organisms that assemble extracellular amyloid or cellulosic materials. Graphical abstract Solid-state NMR was used to quantitatively track the isolation of the insoluble amyloid-associated ECM from uropathogenic E. coli and understand the role of Congo red in purification protocols.

Drug Resistance Patterns of Escherichia coli in Ethiopia: A Meta-Analysis.

PubMed

Tuem, Kald Beshir; Gebre, Abadi Kahsu; Atey, Tesfay Mehari; Bitew, Helen; Yimer, Ebrahim M; Berhe, Derbew Fikadu

2018-01-01

Antimicrobial drug resistance is a global threat for treatment of infectious diseases and costs life and money and threatens health delivery system's effectiveness. The resistance of E. coli to frequently utilized antimicrobial drugs is becoming a major challenge in Ethiopia. However, there is no inclusive countrywide study. Therefore, this study intended to assess the prevalence of E. coli resistance and antimicrobial-specific resistance pattern among E. coli clinical isolates in Ethiopia. Articles were retrieved from PubMed, Embase, and grey literature from 2007 to 2017. The main outcome measures were overall E. coli and drug-specific resistance patterns. A random-effects model was used to determine pooled prevalence with 95% confidence interval (CI), using DerSimonian and Laird method. In addition, subgroup analysis was conducted to improve the outcome. The study bias was assessed by Begg's funnel plot. This study was registered in PROSPERO as follows: PROSPERO 2017: CRD42017070106. Of 164 articles retrieved, 35 articles were included. A total of 19,235 study samples participated in the studies and 2,635 E. coli strains were isolated. Overall, E. coli antibacterial resistance was 45.38% (95% confidence interval (CI): 33.50 to 57.27). The resistance pattern ranges from 62.55% in Addis Ababa to 27.51% in Tigray region. The highest resistance of E. coli reported was to ampicillin (83.81%) and amoxicillin (75.79%), whereas only 13.55% of E. coli isolates showed resistance to nitrofurantoin. E. coli antimicrobial resistance remains high with disparities observed among regions. The bacterium was found to be highly resistant to aminopenicillins. The finding implies the need for effective prevention strategies for the E. coli drug resistance and calls for multifaceted approaches with full involvement of all stakeholders.

Extended-spectrum-β-lactamase-producing Escherichia coli as a cause of pediatric infections: report of a neonatal intensive care unit outbreak due to a CTX-M-14-producing strain.

PubMed

Oteo, Jesús; Cercenado, Emilia; Fernández-Romero, Sara; Saéz, David; Padilla, Belén; Zamora, Elena; Cuevas, Oscar; Bautista, Verónica; Campos, José

2012-01-01

Little information is available about pediatric infections caused by extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli. We characterized an outbreak caused by a CTX-M-14-producing E. coli isolate in a neonatal intensive care unit (NICU) and studied other infections caused by ESBL-producing E. coli in non-NICU pediatric units. All children ≤4 years old who were infected or colonized by ESBL-producing E. coli isolates between January 2009 and September 2010 were included. Molecular epidemiology was studied by phylogroup analysis, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing. Antibiotic resistance genes were analyzed by PCR and sequencing. Plasmids were studied by PFGE with S1 nuclease digestion and by incompatibility group analysis using a PCR-based replicon-typing scheme. Of the ESBL-producing E. coli isolates colonizing or infecting the 30 newborns, identical PFGE results were observed for 21 (70%) isolates, which were classified as CTX-M-14-producing E. coli of ST23 phylogroup A. bla(CTX-M-14a) was linked to ISEcp1 and was carried on an ∼80-bp IncK plasmid. A smaller ongoing outbreak due to SHV-12-producing ST131 E. coli was also identified in the same NICU. Fifteen additional infections with ESBL-producing E. coli were identified in non-NICU pediatric units, but none was caused by the CTX-M-14-producing E. coli epidemic clone. Overall, CTX-M-14 (71.1%), CTX-M-15 (13.3%), and SHV-12 (13.3%) were the most important ESBLs causing pediatric infections in this study. Infections of newborns with CTX-M-14-producing E. coli were caused by both clonal and nonclonal isolates.

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.

Bacterial Expression of a Single-Chain Variable Fragment (scFv) Antibody against Ganoderic Acid A: A Cost-Effective Approach for Quantitative Analysis Using the scFv-Based Enzyme-Linked Immunosorbent Assay.

PubMed

Yusakul, Gorawit; Nuntawong, Poomraphie; Sakamoto, Seiichi; Ratnatilaka Na Bhuket, Pahweenvaj; Kohno, Toshitaka; Kikkawa, Nao; Rojsitthisak, Pornchai; Shimizu, Kuniyoshi; Tanaka, Hiroyuki; Morimoto, Satoshi

2017-01-01

Due to the highly specific binding between an antibody and its target, superior analytical performances was obtained by immunoassays for phytochemical analysis over conventional chromatographic techniques. Here, we describe a simple method for producing a functional single-chain variable fragment (scFv) antibody against ganoderic acid A (GAA), a pharmacologically active metabolite from Ganoderma lingzhi. The Escherichia coli BL21(DE3) strain produced a large amount of anti-GAA scFv. However, in vitro refolding steps, which partially recovered the reactivity of the scFv, were required. Interestingly, the functional scFv was expressed as a soluble and active form in the cytoplasm of an engineered E. coli SHuffle ® strain. Purified anti-GAA scFv, which yielded 2.56 mg from 1 L of culture medium, was obtained from simple and inexpensive procedures for expression and purification. The anti-GAA scFv-based indirect competitive enzyme-linked immunosorbent assay (icELISA) exhibited high sensitivity (linearity: 0.078-1.25 µg/mL) with precision (CV: ≤6.20%) and reliability (recovery: 100.1-101.8%) for GAA determination. In summary, the approach described here is an inexpensive, simple, and efficient expression system that extends the application of anti-GAA scFv-based immunoassays. In addition, when in vitro refolding steps can be skipped, the cost and complexity of scFv antibody production can be minimized.

Reactive oxygen species in plasma against E. coli cells survival rate

NASA Astrophysics Data System (ADS)

Zhou, Ren-Wu; Zhang, Xian-Hui; Zong, Zi-Chao; Li, Jun-Xiong; Yang, Zhou-Bin; Liu, Dong-Ping; Yang, Si-Ze

2015-08-01

In this paper, we report on the contrastive analysis of inactivation efficiency of E. coli cells in solution with different disinfection methods. Compared with the hydrogen peroxide solution and the ozone gas, the atmospheric-pressure He plasma can completely kill the E. coli cells in the shortest time. The inactivation efficiency of E. coli cells in solution can be well described by using the chemical reaction rate model. X-ray photoelectron spectroscopy (XPS) analysis shows that the C-O or C=O content of the inactivated E. coli cell surface by plasma is predominantly increased, indicating the quantity of oxygen-containing species in plasma is more than those of two other methods, and then the C-C or C-H bonds can be broken, leading to the etching of organic compounds. Analysis also indicates that plasma-generated species can play a crucial role in the inactivation process by their direct reactions or the decompositions of reactive species, such as ozone into OH radicals in water, then reacting with E. coli cells. Project supported by the Natural Science Foundation of Fujian Province, China (Grant No. 2014J01025), the National Natural Science Foundation of China (Grant No. 11275261), and the Funds from the Fujian Provincial Key Laboratory for Plasma and Magnetic Resonance, China.

Safe, rapid, and sensitive method of quantitating and distinguishing among Shiga toxins in complex media and human serum

USDA-ARS?s Scientific Manuscript database

Shiga toxins are primarily responsible for the virulence associated with Shiga toxin producing Escherichia coli (STEC) infections. The expression of the Shiga toxins is controlled by a phage that infects the host. More than one phage can infect a host and the host can inactivate infecting phages. T...

Detection and quantitation of Escherichia coli O157:H7 in raw milk by direct qPCR

USDA-ARS?s Scientific Manuscript database

There has been a shift in US food consumption patterns toward unprocessed foods perceived to be healthier. Part of this trend is increasing consumption of raw, unpasteurized milk. Raw milk can be a source of pathogens that cause serious or fatal illness, particularly in immuno-compromised individ...

Comparative multi-omics systems analysis of Escherichia coli strains B and K-12.

PubMed

Yoon, Sung Ho; Han, Mee-Jung; Jeong, Haeyoung; Lee, Choong Hoon; Xia, Xiao-Xia; Lee, Dae-Hee; Shim, Ji Hoon; Lee, Sang Yup; Oh, Tae Kwang; Kim, Jihyun F

2012-05-25

Elucidation of a genotype-phenotype relationship is critical to understand an organism at the whole-system level. Here, we demonstrate that comparative analyses of multi-omics data combined with a computational modeling approach provide a framework for elucidating the phenotypic characteristics of organisms whose genomes are sequenced. We present a comprehensive analysis of genome-wide measurements incorporating multifaceted holistic data - genome, transcriptome, proteome, and phenome - to determine the differences between Escherichia coli B and K-12 strains. A genome-scale metabolic network of E. coli B was reconstructed and used to identify genetic bases of the phenotypes unique to B compared with K-12 through in silico complementation testing. This systems analysis revealed that E. coli B is well-suited for production of recombinant proteins due to a greater capacity for amino acid biosynthesis, fewer proteases, and lack of flagella. Furthermore, E. coli B has an additional type II secretion system and a different cell wall and outer membrane composition predicted to be more favorable for protein secretion. In contrast, E. coli K-12 showed a higher expression of heat shock genes and was less susceptible to certain stress conditions. This integrative systems approach provides a high-resolution system-wide view and insights into why two closely related strains of E. coli, B and K-12, manifest distinct phenotypes. Therefore, systematic understanding of cellular physiology and metabolism of the strains is essential not only to determine culture conditions but also to design recombinant hosts.

Comparative multi-omics systems analysis of Escherichia coli strains B and K-12

PubMed Central

2012-01-01

Background Elucidation of a genotype-phenotype relationship is critical to understand an organism at the whole-system level. Here, we demonstrate that comparative analyses of multi-omics data combined with a computational modeling approach provide a framework for elucidating the phenotypic characteristics of organisms whose genomes are sequenced. Results We present a comprehensive analysis of genome-wide measurements incorporating multifaceted holistic data - genome, transcriptome, proteome, and phenome - to determine the differences between Escherichia coli B and K-12 strains. A genome-scale metabolic network of E. coli B was reconstructed and used to identify genetic bases of the phenotypes unique to B compared with K-12 through in silico complementation testing. This systems analysis revealed that E. coli B is well-suited for production of recombinant proteins due to a greater capacity for amino acid biosynthesis, fewer proteases, and lack of flagella. Furthermore, E. coli B has an additional type II secretion system and a different cell wall and outer membrane composition predicted to be more favorable for protein secretion. In contrast, E. coli K-12 showed a higher expression of heat shock genes and was less susceptible to certain stress conditions. Conclusions This integrative systems approach provides a high-resolution system-wide view and insights into why two closely related strains of E. coli, B and K-12, manifest distinct phenotypes. Therefore, systematic understanding of cellular physiology and metabolism of the strains is essential not only to determine culture conditions but also to design recombinant hosts. PMID:22632713

Temporal variations of Escherichia coli concentrations in a large Midwestern river

USGS Publications Warehouse

Schilling, K.E.; Zhang, Y.-K.; Hill, D.R.; Jones, C.S.; Wolter, C.F.

2009-01-01

The Raccoon River used by the Des Moines Water Works to serve more than 400,000 people in central Iowa is threatened by contamination from Escherichia coli bacteria from point and nonpoint sources. The 9389 km2 watershed is highly agricultural, with 73% of the land in row crop production and widespread animal production. Results from 2155 grab samples from 1997 to 2005 for E. coli analysis were examined for temporal variations. E. coli concentrations were found to vary across years, seasons, and flow conditions, with a 9-year mean value of 1156 most probable number (MPN)/100 ml. Monthly concentrations exhibited clear seasonality with highest values in May through July. Although E. coli concentrations were higher during periods of greater discharge, the relation of log E. coli to log discharge was not particularly strong (r2 = 0.35). The variogram of E. coli concentrations showed temporal correlation within a span of 4 days suggesting that concentrations measured on 1 day may be related in time to concentrations measured up to 3 days later and beyond 4 days the concentrations vary randomly. The spectral analysis of the time series of E. coli was also carried out and was fitted well with the spectrum of an exponential covariance function. Deciphering temporal patterns and correlation of E. coli bacteria in streams may be useful for developing future monitoring strategies to track concentration patterns and loads. ?? 2008 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2016-01-01

ABSTRACT The global spread of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli (ESBL-E. coli) has largely been driven by the pandemic sequence type 131 (ST131). This study aimed to determine the molecular epidemiology of their spread in two Asian countries with contrasting prevalence. We conducted whole-genome sequencing (WGS) of ESBL-E. coli ST131 strains collected prospectively from Nepal and Japan, two countries in Asia with a high and low prevalence of ESBL-E. coli, respectively. We also systematically compared these genomes with those reported from other regions using publicly available WGS data for E. coli ST131 strains. Further, we conducted phylogenetic analysis of these isolates and all genome sequence data for ST131 strains to determine sequence diversity. One hundred five unique ESBL-E. coli isolates from Nepal (February 2013 to July 2013) and 76 isolates from Japan (October 2013 to September 2014) were included. Of these isolates, 54 (51%) isolates from Nepal and 11 (14%) isolates from Japan were identified as ST131 by WGS. Phylogenetic analysis based on WGS suggested that the majority of ESBL-E. coli ST131 isolates from Nepal clustered together, whereas those from Japan were more diverse. Half of the ESBL-E. coli ST131 isolates from Japan belonged to virotype C, whereas half of the isolates from Nepal belonged to a virotype other than virotype A, B, C, D, or E (A/B/C/D/E). The dominant sublineage of E. coli ST131 was H30Rx, which was most prominent in ESBL-E. coli ST131 isolates from Nepal. Our results revealed distinct phylogenetic characteristics of ESBL-E. coli ST131 spread in the two geographical areas of Asia, indicating the involvement of multiple factors in its local spread in each region. IMPORTANCE The global spread of ESBL-E. coli has been driven in large part by pandemic sequence type 131 (ST131). A recent study suggested that, within E. coli ST131, certain sublineages have disseminated worldwide with little association with their geographical origin, highlighting the complexity of the epidemiology of this pandemic clone. ST131 bacteria have also been classified into four virotypes based on the distribution of certain virulence genes. Information on virotype distribution in Asian ST131 strains is limited. We conducted whole-genome sequencing of ESBL-E. coli ST131 strains collected in Nepal and Japan, two Asian countries with a high and low prevalence of ESBL-E. coli, respectively. We systematically compared these ST131 genomes with those reported from other regions to gain insights into the molecular epidemiology of their spread and found the distinct phylogenetic characteristics of the spread of ESBL-E. coli ST131 in these two geographical areas of Asia. PMID:27830191

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.

Use of Bayesian networks in predicting contamination of drinking water with E. coli in rural Vietnam.

PubMed

Hall, David C; Le, Quynh B

2017-06-01

More than 70 million Vietnamese rely on small-scale farming for some form of household income. Water on many of those farms is contaminated with waste, including animal manure, partly due to non-sustainable waste management. This increases the risk of water-related zoonotic disease transmission. The purpose of this research was to examine the impact of various demographic and management factors on the likelihood of finding Escherichia coli in drinking water sourced from wells and rainwater on farms in Vietnam. A Bayesian Belief Network (BBN) was designed to describe association between various deterministic and probabilistic variables gathered from 600 small-scale integrated (SSI) farmers in Vietnam. The variables relate to E. coli content of their drinking water sourced on-farm from wells and rainwater, and stored in on-farm large vessels, including concrete water tanks. The BBN was developed using the Netica software tool; the model was calibrated and goodness of fit examined using concordance of predictability. Sensitivity analysis of the model revealed that choice variables, including engagement in mitigation of water contamination and livestock management activities, were particularly likely to influence endpoint values, reflecting the highly variable and impactful nature of preferences, attitudes and beliefs relating to mitigation strategies. Quantitative variables including numbers of livestock (particularly chickens) and income also had a high impact. The highest concordance (62%) was achieved with the BBN reported in this paper. This BBN model of SSI farming in Vietnam is helpful in understanding the complexity of small-scale agriculture and how various factors work in concert to influence contamination of on-farm drinking water as indicated by the presence of E. coli. The model will also be useful for identifying and estimating the impact of policy options such as improved delivery of clean water management training for rural areas, particularly where such analysis is combined with other analytical and policy tools. With appropriate knowledge translation, the model results will be particularly useful in helping SSI farmers understand their options for engaging in public health mitigation strategies addressing clean water that do not significantly disrupt their agriculture-based livelihoods. © The Author 2017. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Genotypic analysis of virulence genes and antimicrobial profile of diarrheagenic Escherichia coli isolated from diseased lambs in Iran.

PubMed

Ghanbarpour, Reza; Askari, Nasrin; Ghorbanpour, Masoud; Tahamtan, Yahya; Mashayekhi, Khoobyar; Afsharipour, Narjes; Darijani, Nasim

2017-03-01

The aim of the present study was to determine the analysis of virulence genes and antimicrobial profile of diarrheagenic Escherichia coli isolated from diseased lambs. Two hundred ninety E. coli isolates were recovered from 300 rectal swabs of diarrheic lambs and were confirmed by biochemical tests. The pathotype determination was done according to the presence of genes including f5, f41, LTI, STI, bfp, ipaH, stx 1 , stx 2 , eae, ehlyA, cnf 1 , cnf 2 , cdIII, cdIV, and f17 by PCR method. Sixty-six isolates (23.72%) possessed the STI gene and categorized into entrotoxigenic E. coli (ETEC). Nine isolates (3.1%) and five isolates (1.72%) were positive for the cnf1 and cnf2 genes which categorized into necrotoxic E. coli (NTEC). Hundred and seventeen isolates (40.34%) harbored stx 1 and/or stx 2 and classified as Shiga toxin-producing E. coli (STEC). Thirteen isolates (4.48%) were assigned to atypical entropathogenic E. coli (aEPEC) and possessed eae gene. Two isolates (0.68%) were positive for ipaH gene and were assigned to entroinvasive E. coli (EIEC). Statistical analysis showed a specific association between eae gene and STEC pathotype (P < 0.0001). The most prevalent resistance was observed against lincomycin (96.5%) and the lowest resistance was against kanamycine (56.02%), respectively. The high prevalence of STEC and ETEC indicates that diarrheic lambs represent an important reservoir for humans. ETEC may play an important role for frequent occurrence of diarrhea in lambs observed in this region. Due to high antibiotic resistance, appropriate control should be implemented in veterinary medicine to curb the development of novel resistant isolates.

Expression of codon-optmized phosphoenolpyruvate carboxylase gene from Glaciecola sp. HTCC2999 in Escherichia coli and its application for C4 chemical production.

PubMed

Park, Soohyun; Pack, Seung Pil; Lee, Jinwon

2012-08-01

We examined the expression of the phosphoenolpyruvate carboxylase (PEPC) gene from marine bacteria in Escherichia coli using codon optimization. The codon-optimized PEPC gene was expressed in the E. coli K-12 strain W3110. SDS-PAGE analysis revealed that the codon-optimized PEPC gene was only expressed in E. coli, and measurement of enzyme activity indicated the highest PEPC activity in the E. coli SGJS112 strain that contained the codon-optimized PEPC gene. In fermentation assays, the E. coli SGJS112 produced the highest yield of oxaloacetate using glucose as the source and produced a 20-times increase in the yield of malate compared to the control. We concluded that the codon optimization enabled E. coli to express the PEPC gene derived from the Glaciecola sp. HTCC2999. Also, the expressed protein exhibited an enzymatic activity similar to that of E. coli PEPC and increased the yield of oxaloacetate and malate in an E. coli system.

75 FR 32295 - Expedited Approval of Alternative Test Procedures for the Analysis of Contaminants Under the Safe...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-08

... Identification of Coliform Bacteria and Escherichia coli in Finished Waters, January 2007, Version 1.1... Membrane Filter Test Method for Detection and Identification of Coliform Bacteria and Escherichia coli in... Detection and Identification of Coliform Bacteria and Escherichia coli in Finished Waters. November, 2000...

Identification, structural characterisation and expression analysis of a defensin gene from the tiger beetle Calomera littoralis (Coleoptera: Cicindelidae).

PubMed

Rodríguez-García, María Juliana; García-Reina, Andrés; Machado, Vilmar; Galián, José

2016-09-01

In this study, a defensin gene (Clit-Def) has been characterised in the tiger beetle Calomera littoralis for the first time. Bioinformatic analysis showed that the gene has an open reading frame of 246bp that contains a 46 amino acid mature peptide. The phylogenetic analysis showed a high variability in the coleopteran defensins analysed. The Clit-Def mature peptide has the features to be involved in the antimicrobial function: a predicted cationic isoelectric point of 8.94, six cysteine residues that form three disulfide bonds, and the typical cysteine-stabilized α-helix β-sheet (CSαβ) structural fold. Real time quantitative PCR analysis showed that Clit-Def was upregulated in the different body parts analysed after infection with lipopolysaccharides of Escherichia coli, and also indicated that has an expression peak at 12h post infection. The expression patterns of Clit-Def suggest that this gene plays important roles in the humoral system in the adephagan beetle Calomera littoralis. Copyright © 2016 Elsevier B.V. All rights reserved.

The Escherichia coli Proteome: Past, Present, and Future Prospects†

PubMed Central

Han, Mee-Jung; Lee, Sang Yup

2006-01-01

Proteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects. PMID:16760308

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

PubMed Central

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

1984-01-01

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

Rates of spontaneous mutation in an archaeon from geothermal environments.

PubMed Central

Jacobs, K L; Grogan, D W

1997-01-01

To estimate the efficacy of mechanisms which may prevent or repair thermal damage to DNA in thermophilic archaea, a quantitative assay of forward mutation at extremely high temperature was developed for Sulfolobus acidocaldarius, based on the selection of pyrimidine-requiring mutants resistant to 5-fluoro-orotic acid. Maximum-likelihood analysis of spontaneous mutant distributions in wild-type cultures yielded maximal estimates of (2.8 +/- 0.7) x 10(-7) and (1.5 +/- 0.6) x 10(-7) mutational events per cell per division cycle for the pyrE and pyrF loci, respectively. To our knowledge, these results provide the first accurate measurement of the genetic fidelity maintained by archaea that populate geothermal environments. The measured rates of forward mutation at the pyrE and pyrF loci in S. acidocaldarius are close to corresponding rates reported for protein-encoding genes of Escherichia coli. The normal rate of spontaneous mutation in E. coli at 37 degrees C is known to require the functioning of several enzyme systems that repair spontaneous damage in DNA. Our results provide indirect evidence that S. acidocaldarius has cellular mechanisms, as yet unidentified, which effectively compensate for the higher chemical instability of DNA at the temperatures and pHs that prevail within growing Sulfolobus cells. PMID:9150227

The role of MatP, ZapA and ZapB in chromosomal organization and dynamics in Escherichia coli

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

Mannik, Jaana; Castillo, Daniel E.; Yang, Da

Despite extensive research over several decades, a comprehensive view of how the Escherichia coli chromosome is organized within the nucleoid, and how two daughter chromosomes segregate has yet to emerge. Here we investigate the role of the MatP, ZapA and ZapB proteins in organizing the replication terminus (Ter) region and in the chromosomal segregation process. Quantitative image analysis of the fluorescently labeled Ter region shows that the replication terminus attaches to the divisome in a single segment along the perimeter of the cell in a MatP, ZapA and ZapB-dependent manner. The attachment does not significantly affect the bulk chromosome segregationmore » in slow growth conditions. With or without the attachment, two chromosomal masses separate from each other at a speed comparable to the cell growth. The separation starts even before the replication terminus region positions itself at the center of the nucleoid. Modeling of the segregation based on conformational entropy correctly predicts the positioning of the replication terminus region within the nucleoid. Furthermore, the model produces a distinctly different chromosomal density distribution than the experiment, indicating that the conformational entropy plays a limited role in segregating the chromosomes in the late stages of replication.« less

Enrichment and Detection of Escherichia coli O157:H7 from Water Samples Using an Antibody Modified Microfluidic Chip

PubMed Central

Dharmasiri, Udara; Witek, Małgorzata A.; Adams, Andre A.; Osiri, John K.; Hupert, Mateusz L.; Bianchi, Thomas S.; Roelke, Daniel L.; Soper, Steven A.

2010-01-01

Low abundant (<100 cells mL-1) E. coli O157:H7 cells were isolated and enriched from environmental water samples using a microfluidic chip. The poly(methylmethacrylate), PMMA, chip contained 8 devices each equipped with 16 curvilinear high aspect ratio channels that were covalently decorated with polyclonal anti-O157 antibodies (pAb) and could search for rare cells through a pAb mediated process. The chip could process independently 8 different samples or one sample using 8 different parallel inputs to increase volume processing throughput. After cell enrichment, cells were released and enumerated using bench top real-time quantitative PCR, targeting genes which effectively discriminated the O157:H7 serotype from other non-pathogenic bacteria. The recovery of target cells from water samples was determined to be ~72%, and the limit-of-detection was found to be 6 colony forming units (cfu) using the slt1 gene as a reporter. We subsequently performed analysis of lake and waste water samples. The simplicity in manufacturing and ease of operation makes this device attractive for the selection of pathogenic species from a variety of water supplies suspected of containing bacterial pathogens at extremely low frequencies. PMID:20218574

Comparative study on the epidemiological aspects of enterohemorrhagic Escherichia coli infections between Korea and Japan, 2006 to 2010.

PubMed

Lee, Won-Chang; Kwon, Young Hwan

2016-05-01

To compare the epidemiological aspects of enterohemorrhagic Escherichia coli (EHEC) between Korea and Japan by analyzing the current state of EHEC infection outbreaks and related risk factors. We investigated the epidemiological aspects of EHEC infection cases between Korea and Japan from 2006 to 2010. The following factors were analyzed: national prevalence rate (PR), regional prevalence rate, epidemic aspects (i.e., Cases related to gender), male to female morbidity ratio, age, and seasonal distribution. In total, there were 254 cases of EHEC with an average PR of 0.11 per 100,000 populations in Korea from 2006 to 2010. During the same period in Japan, there were 20,883 cases of EHEC with an average PR of 3.26 per 100,000 populations. The PR in Japan was significantly higher than that in Korea (p < 0.01). In both countries, more females than males had EHEC infections, with the highest incidence of infections (> 50%) observed for individuals younger than 9 years. EHEC is an emerging zoonosis and may be caused by consumption of raw or undercooked meat products from ruminants. This study provides a quantitative analysis of the epidemiological aspects and risk factors of EHEC infections in Korea and Japan and will provide insight on effective future strategies to reduce these infections.

The role of MatP, ZapA and ZapB in chromosomal organization and dynamics in Escherichia coli

DOE PAGES

Mannik, Jaana; Castillo, Daniel E.; Yang, Da; ...

2016-01-13

Despite extensive research over several decades, a comprehensive view of how the Escherichia coli chromosome is organized within the nucleoid, and how two daughter chromosomes segregate has yet to emerge. Here we investigate the role of the MatP, ZapA and ZapB proteins in organizing the replication terminus (Ter) region and in the chromosomal segregation process. Quantitative image analysis of the fluorescently labeled Ter region shows that the replication terminus attaches to the divisome in a single segment along the perimeter of the cell in a MatP, ZapA and ZapB-dependent manner. The attachment does not significantly affect the bulk chromosome segregationmore » in slow growth conditions. With or without the attachment, two chromosomal masses separate from each other at a speed comparable to the cell growth. The separation starts even before the replication terminus region positions itself at the center of the nucleoid. Modeling of the segregation based on conformational entropy correctly predicts the positioning of the replication terminus region within the nucleoid. Furthermore, the model produces a distinctly different chromosomal density distribution than the experiment, indicating that the conformational entropy plays a limited role in segregating the chromosomes in the late stages of replication.« less

The effect of metal microstructure on the initial attachment of Escherichia coli to 1010 carbon steel.

PubMed

Javed, M A; Stoddart, P R; McArthur, S L; Wade, S A

2013-09-01

Metallurgical features have been shown to play an important role in the attachment of microorganisms to metal surfaces. In the present study, the influence of the microstructure of as-received (AR) and heat-treated (HT) 1010 carbon steel on the initial attachment of bacteria was investigated. Heat treatment was carried out with the aim of increasing the grain size of the carbon steel coupons. Mirror-polished carbon steel coupons were immersed in a minimal medium inoculated with Escherichia coli (ATCC 25922) to investigate the early (15, 30 and 60 min) and relatively longer-term (4 h) stages of bacterial attachment. The results showed preferential colonisation of bacteria on the grain boundaries of the steel coupons. The bacterial attachment to AR steel coupons was relatively uniform compared to the HT steel coupons where an increased number of localised aggregates of bacteria were found. Quantitative analysis showed that the ratio of the total number of isolated (i.e., single) bacteria to the number of bacteria in aggregates was significantly higher on the AR coupons than the HT coupons. Longer-term immersion studies showed production of extracellular polymeric substances by the bacteria and corrosion at the grain boundaries on both types of steel coupon tested.

The Escherichia coli cAMP receptor protein bound at a single target can activate transcription initiation at divergent promoters: a systematic study that exploits new promoter probe plasmids.

PubMed Central

El-Robh, Mohamed Samir; Busby, Stephen J W

2002-01-01

We report the first detailed quantitative study of divergent promoters dependent on the Escherichia coli cAMP receptor protein (CRP), a factor known to activate transcription initiation at target promoters by making direct interactions with the RNA polymerase holoenzyme. In this work, we show that CRP bound at a single target site is able to activate transcription at two divergently organized promoters. Experiments using promoter probe plasmids, designed to study divergent promoters in vivo and in vitro, show that the divergent promoters function independently. Further in vitro experiments show that two holo RNA polymerase molecules cannot be accommodated simultaneously at the divergent promoters. PMID:12350222

Survival of enteric pathogens in common beverages: an in vitro study.

PubMed

Sheth, N K; Wisniewski, T R; Franson, T R

1988-06-01

This in vitro study was undertaken to determine the potential for survival of enteric pathogens in common drinking beverages. Three carbonated soft drinks, two alcoholic beverages, skim milk, and water were inoculated with Salmonella, Shigella, and enterotoxigenic Escherichia coli, and quantitative counts were performed over 2 days. Our studies showed poorest survival of all three organisms in wine, and greatest growth in milk and water. Beer and cola allowed survival of small numbers of Salmonella and E. coli at 48 h, whereas sour mix and diet cola were sterile by 48 h. Survival features may correlate with pH of the beverages. These observations may be useful in guiding travellers for appropriate beverage consumption while visiting areas endemic for "traveller's diarrhea."

Analysis of the SDS-PAGE patterns of outer membrane proteins from Escherichia coli strains that have lost the ability to form K1 antigen and varied in the susceptibility to normal human serum.

PubMed

Cisowska, Agnieszka; Bugla-Płoskońska, Gabriela

2014-01-01

We used SDS-polyacrylamide gel electrophoresis to investigate the outer membrane proteins (OMPs) band composition of 19 Escherichia coli K1 strains that have spontaneously lost the ability to form K1 polysaccharide capsule (E. coli K1-) and demonstrated different degrees of susceptibility to the bactericidal action of normal human serum. Presented results showed that there were differences between E. coli K1- strains in OMPs expressing capacity. The analysis performed on OMPs has not revealed a direct association between the different OMPs band composition and the susceptibility of these strains to the serum.

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.

High Frequency of Diarrheagenic Escherichia coli in HIV-Infected Patients and Patients with Thalassemia in Kerman, Iran.

PubMed

Alizade, Hesam; Sharifi, Hamid; Naderi, Zahedeh; Ghanbarpour, Reza; Bamorovat, Mehdi; Aflatoonian, Mohammad Reza

This study was conducted on patients with thalassemia and HIV-infected patients to determine the frequency of diarrheagenic Escherichia coli in Kerman, Iran. We analyzed 68 and 49 E coli isolates isolated from healthy fecal samples of patients with thalassemia and HIV-infected patients, respectively. The E coli isolates were studied using a multiplex polymerase chain reaction to identify the enterotoxigenic E coli (ETEC), enterohemorrhagic E coli (EHEC), and enteropathogenic E coli (EPEC) groups. Statistical analysis was carried out to determine the correlation of diarrheagenic E coli between HIV-infected patients and patients with thalassemia using Stata 11.2 software. The frequency of having at least 1 diarrheagenic E coli was more common in patients with thalassemia (67.64%) than in HIV-infected patients (57.14%; P = .25), including ETEC (67.64% versus 57.14%), EHEC (33.82% versus 26.53%), and EPEC (19.11% versus 16.32%). The results of this study indicate that ETEC, EHEC, and EPEC pathotypes are widespread among diarrheagenic E coli isolates in patients with thalassemia and HIV-infected patients.

Application of Quantitative Microbial Risk Assessment to analyze the public health risk from poor drinking water quality in a low income area in Accra, Ghana.

PubMed

Machdar, E; van der Steen, N P; Raschid-Sally, L; Lens, P N L

2013-04-01

In Accra, Ghana, a majority of inhabitants lives in over-crowded areas with limited access to piped water supply, which is often also intermittent. This study assessed in a densely populated area the risk from microbial contamination of various sources of drinking water, by conducting a Quantitative Microbiological Risk Assessment (QMRA) to estimate the risk to human health from microorganism exposure and dose-response relationships. Furthermore the cost-effectiveness in reducing the disease burden through targeted interventions was evaluated. Five risk pathways for drinking water were identified through a survey (110 families), namely household storage, private yard taps, communal taps, communal wells and water sachets. Samples from each source were analyzed for Escherichia coli and Ascaris contamination. Published ratios between E. coli and other pathogens were used for the QMRA and disease burden calculations. The major part of the burden of disease originated from E. coli O157:H7 (78%) and the least important contributor was Cryptosporidium (0.01%). Other pathogens contributed 16% (Campylobacter), 5% (Rotavirus) and 0.3% (Ascaris). The sum of the disease burden of these pathogens was 0.5 DALYs per person per year, which is much higher than the WHO reference level. The major contamination pathway was found to be household storage. Disinfection of water at household level was the most cost-effective intervention (<5 USD/DALY-averted) together with hygiene education. Water supply network improvements were significantly less cost-effective. Copyright © 2013 Elsevier B.V. All rights reserved.

Microbial Quality, Safety, and Pathogen Detection by Using Quantitative PCR of Raw Salad Vegetables Sold in Dhanbad City, India.

PubMed

Mritunjay, Sujeet K; Kumar, Vipin

2017-01-01

Consumption of ready-to-eat fresh vegetables has increased worldwide, with a consequent increase in outbreaks caused by foodborne pathogens. In the Indian subcontinent, raw fresh vegetables are usually consumed without washing or other decontamination procedures, thereby leading to new food safety threats. In this study, the microbiological quality and pathogenic profile of raw salad vegetables was evaluated through standard protocols. In total, 480 samples (60 each of eight different salad vegetables) of cucumber, tomato, carrot, coriander, cabbage, beetroot, radish, and spinach were collected from different locations in Dhanbad, a city famous for its coal fields and often called the "Coal Capital of India." The samples were analyzed for total plate count, total coliforms, Escherichia coli , E. coli O157:H7, Listeria monocytogenes , and Salmonella spp. Incidences of pathogens were detected through quantitative PCR subsequent to isolation. Results showed that 46.7% (for total plate counts) and 30% (for total coliforms) of samples were unacceptable for consumption per the Food Safety and Standards Authority of India. Pathogenic microorganisms were detected in 3.7% of total samples. E. coli O157:H7 was detected in three samples of spinach (2) and beetroot ( 1 ); L. monocytogenes was detected in 14 samples of spinach ( 8 ), tomato ( 3 ), cucumber ( 2 ), and radish ( 1 ); and Salmonella spp. were detected in 16 samples of spinach ( 7 ), tomato ( 3 ), beetroot ( 2 ), cucumber ( 2 ), carrot ( 1 ), and radish ( 1 ). Pathogens were not detected in any of the cabbage and coriander samples.

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.

Occurrence of Diarrheagenic Virulence Genes and Genetic Diversity in Escherichia coli Isolates from Fecal Material of Various Avian Hosts in British Columbia, Canada

PubMed Central

Mazumder, Asit

2014-01-01

Contamination of surface water by fecal microorganisms originating from human and nonhuman sources is a public health concern. In the present study, Escherichia coli isolates (n = 412) from the feces of various avian host sources were screened for various virulence genes: stx1 and stx2 (Shiga toxin-producing E. coli [STEC]), eae (enteropathogenic E. coli [EPEC]), est-h, est-p, and elt (encoding heat-stable toxin [ST] variants STh and STp and heat-labile toxin [LT], respectively) (enterotoxigenic E. coli [ETEC]), and ipaH (enteroinvasive E. coli [EIEC]). None of the isolates were found to be positive for stx1, while 23% (n = 93) were positive for only stx2, representing STEC, and 15% (n = 63) were positive for only eae, representing EPEC. In addition, five strains obtained from pheasant were positive for both stx2 and eae and were confirmed as non-O157 by using an E. coli O157 rfb (rfbO157) TaqMan assay. Isolates positive for the virulence genes associated with ETEC and EIEC were not detected in any of the hosts. The repetitive element palindromic PCR (rep-PCR) fingerprint analysis identified 143 unique fingerprints, with an overall Shannon diversity index of 2.36. Multivariate analysis of variance (MANOVA) showed that the majority of the STEC and EPEC isolates were genotypically distinct from nonpathogenic E. coli and clustered independently. MANOVA analysis also revealed spatial variation among the E. coli isolates, since the majority of the isolates clustered according to the sampling locations. Although the presence of virulence genes alone cannot be used to determine the pathogenicity of strains, results from this study show that potentially pathogenic STEC and EPEC strains can be found in some of the avian hosts studied and may contaminate surface water and potentially impact human health. PMID:24441159

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

PubMed

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

2007-09-01

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

Dynamics of Escherichia coli Virulence Factors in Dairy Herds and Farm Environments in a Longitudinal Study in the United States

PubMed Central

Lambertini, Elisabetta; Karns, Jeffrey S.; Van Kessel, Jo Ann S.; Cao, Huilin; Schukken, Ynte H.; Wolfgang, David R.; Smith, Julia M.

2015-01-01

Pathogenic Escherichia coli or its associated virulence factors have been frequently detected in dairy cow manure, milk, and dairy farm environments. However, it is unclear what the long-term dynamics of E. coli virulence factors are and which farm compartments act as reservoirs. This study assessed the occurrence and dynamics of four E. coli virulence factors (eae, stx1, stx2, and the gamma allele of the tir gene [γ-tir]) on three U.S. dairy farms. Fecal, manure, water, feed, milk, and milk filter samples were collected from 2004 to 2012. Virulence factors were measured by postenrichment quantitative PCR (qPCR). All factors were detected in most compartments on all farms. Fecal and manure samples showed the highest prevalence, up to 53% for stx and 21% for γ-tir in fecal samples and up to 84% for stx and 44% for γ-tir in manure. Prevalence was low in milk (up to 1.9% for stx and 0.7% for γ-tir). However, 35% of milk filters were positive for stx and 20% were positive for γ-tir. All factors were detected in feed and water. Factor prevalence and levels, expressed as qPCR cycle threshold categories, fluctuated significantly over time, with no clear seasonal signal independent from year-to-year variability. Levels were correlated between fecal and manure samples, and in some cases autocorrelated, but not between manure and milk filters. Shiga toxins were nearly ubiquitous, and 10 to 18% of the lactating cows were potential shedders of E. coli O157 at least once during their time in the herds. E. coli virulence factors appear to persist in many areas of the farms and therefore contribute to transmission dynamics. PMID:25911478

Development of goose- and duck-specific DNA markers to determine sources of Escherichia coli in waterways.

PubMed

Hamilton, Matthew J; Yan, Tao; Sadowsky, Michael J

2006-06-01

The contamination of waterways with fecal material is a persistent threat to public health. Identification of the sources of fecal contamination is a vital component for abatement strategies and for determination of total maximum daily loads. While phenotypic and genotypic techniques have been used to determine potential sources of fecal bacteria in surface waters, most methods require construction of large known-source libraries, and they often fail to adequately differentiate among environmental isolates originating from different animal sources. In this study, we used pooled genomic tester and driver DNAs in suppression subtractive hybridizations to enrich for host source-specific DNA markers for Escherichia coli originating from locally isolated geese. Seven markers were identified. When used as probes in colony hybridization studies, the combined marker DNAs identified 76% of the goose isolates tested and cross-hybridized, on average, with 5% of the human E. coli strains and with less than 10% of the strains obtained from other animal hosts. In addition, the combined probes identified 73% of the duck isolates examined, suggesting that they may be useful for determining the contribution of waterfowl to fecal contamination. However, the hybridization probes reacted mainly with E. coli isolates obtained from geese in the upper midwestern United States, indicating that there is regional specificity of the markers identified. Coupled with high-throughput, automated macro- and microarray screening, these markers may provide a quantitative, cost-effective, and accurate library-independent method for determining the sources of genetically diverse E. coli strains for use in source-tracking studies. However, future efforts to generate DNA markers specific for E. coli must include isolates obtained from geographically diverse animal hosts.

Characteristics of CTX-M Extended-Spectrum β-Lactamase-Producing Escherichia coli Strains Isolated from Multiple Rivers in Southern Taiwan

PubMed Central

Chen, Po-An; Hung, Chih-Hsin; Huang, Ping-Chih; Chen, Jung-Ren; Huang, I-Fei; Chen, Wan-Ling; Chiou, Yee-Hsuan; Hung, Wan-Yu

2016-01-01

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli sequence type ST131 has emerged as the leading cause of community-acquired urinary tract infections and bacteremia worldwide. Whether environmental water is a potential reservoir of these strains remains unclear. River water samples were collected from 40 stations in southern Taiwan from February to August 2014. PCR assay and multilocus sequence typing (MLST) analysis were conducted to determine the CTX-M group and sequence type, respectively. In addition, we identified the seasonal frequency of ESBL-producing E. coli strains and their geographical relationship with runoffs from livestock and poultry farms between February and August 2014. ESBL-producing E. coli accounted for 30% of the 621 E. coli strains isolated from river water in southern Taiwan. ESBL-producing E. coli ST131 was not detected among the isolates. The most commonly detected strain was E. coli CTX-M group 9. Among the 92 isolates selected for MLST analysis, the most common ESBL-producing clonal complexes were ST10 and ST58. The proportion of ESBL-producing E. coli was significantly higher in areas with a lower river pollution index (P = 0.025) and regions with a large number of chickens being raised (P = 0.013). ESBL-producing E. coli strains were commonly isolated from river waters in southern Taiwan. The most commonly isolated ESBL-producing clonal complexes were ST10 and ST58, which were geographically related to chicken farms. ESBL-producing E. coli ST131, the major clone causing community-acquired infections in Taiwan and worldwide, was not detected in river waters. PMID:26773082

Response of Nursery Pigs to a Synbiotic Preparation of Starch and an Anti-Escherichia coli K88 Probiotic ▿

PubMed Central

Krause, D. O.; Bhandari, S. K.; House, J. D.; Nyachoti, C. M.

2010-01-01

Postweaning diarrhea in pigs is frequently caused by enterotoxigenic Escherichia coli K88 (ETEC). The aim of this study was to test the efficacy of E. coli probiotics (PRO) in young pigs challenged with E. coli K88. We also tested the synbiotic interaction with raw potato starch (RPS), which can be used as a prebiotic. Forty 17-day-old weaned piglets were randomly assigned to four treatments: treatment 1, positive-control diet (C), no probiotics or RPS but containing in-feed antibiotics; treatment 2, probiotic (PRO), no feed antibiotics plus a 50:50 mixture of probiotic E. coli strains UM-2 and UM-7; treatment 3, 14% RPS, no antibiotics (RPS); treatment 4, 14% RPS plus a 50:50 mixture of probiotic E. coli strains UM-2 and UM-7, no antibiotics (PRO-RPS). The pigs were challenged with pathogenic E. coli K88 strains on day 7 of the experiment (24-day-old pigs) and euthanized on day 10 of the experiment (35-day-old pigs). Probiotic and pathogenic E. coli strains were enumerated by selective enrichment on antibiotics, and microbial community analysis was conducted using terminal restriction length polymorphism analysis (T-RFLP) of 16S rRNA genes. The combination of raw potato starch and the probiotic had a beneficial effect on piglet growth performance and resulted in a reduction of diarrhea and increased microbial diversity in the gut. We conclude that the use of E. coli probiotic strains against E. coli K88 in the presence of raw potato starch is effective in reducing the negative effects of ETEC in a piglet challenge model. PMID:20952649

Characteristics of CTX-M Extended-Spectrum β-Lactamase-Producing Escherichia coli Strains Isolated from Multiple Rivers in Southern Taiwan.

PubMed

Chen, Po-An; Hung, Chih-Hsin; Huang, Ping-Chih; Chen, Jung-Ren; Huang, I-Fei; Chen, Wan-Ling; Chiou, Yee-Hsuan; Hung, Wan-Yu; Wang, Jiun-Ling; Cheng, Ming-Fang

2016-01-15

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli sequence type ST131 has emerged as the leading cause of community-acquired urinary tract infections and bacteremia worldwide. Whether environmental water is a potential reservoir of these strains remains unclear. River water samples were collected from 40 stations in southern Taiwan from February to August 2014. PCR assay and multilocus sequence typing (MLST) analysis were conducted to determine the CTX-M group and sequence type, respectively. In addition, we identified the seasonal frequency of ESBL-producing E. coli strains and their geographical relationship with runoffs from livestock and poultry farms between February and August 2014. ESBL-producing E. coli accounted for 30% of the 621 E. coli strains isolated from river water in southern Taiwan. ESBL-producing E. coli ST131 was not detected among the isolates. The most commonly detected strain was E. coli CTX-M group 9. Among the 92 isolates selected for MLST analysis, the most common ESBL-producing clonal complexes were ST10 and ST58. The proportion of ESBL-producing E. coli was significantly higher in areas with a lower river pollution index (P = 0.025) and regions with a large number of chickens being raised (P = 0.013). ESBL-producing E. coli strains were commonly isolated from river waters in southern Taiwan. The most commonly isolated ESBL-producing clonal complexes were ST10 and ST58, which were geographically related to chicken farms. ESBL-producing E. coli ST131, the major clone causing community-acquired infections in Taiwan and worldwide, was not detected in river waters. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Structural Analysis of the Dimerization Domain of the Human Estrogen Receptor and a Peptide Inhibitor of Dimerization

DTIC Science & Technology

1998-08-01

communication). Various hER fragments were expressed in Esherichia coli (E. coli ) as glutathione-S-transferace (GST) fusion proteins, separated by...Using an E. coli expression vector, we successfully overexpressed hER[253-341] as a fusion protein with an N-terminal poly-histidine tag (Figure 1A...of hER fused to GST were expressed in E. coli , and they were then separated on SDS PAGE, and then transferred to a blotting membrane. The membrane was

A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins.

PubMed

Burbelo, Peter D; Goldman, Radoslav; Mattson, Thomas L

2005-08-18

Assays detecting human antigen-specific antibodies are medically useful. However, the usefulness of existing simple immunoassay formats is limited by technical considerations such as sera antibodies to contaminants in insufficiently pure antigen, a problem likely exacerbated when antigen panels are screened to obtain clinically useful data. We developed a novel and simple immunoprecipitation technology for identifying clinical sera containing antigen-specific antibodies and for generating quantitative antibody response profiles. This method is based on fusing protein antigens to an enzyme reporter, Renilla luciferase (Ruc), and expressing these fusions in mammalian cells, where mammalian-specific post-translational modifications can be added. After mixing crude extracts, sera and protein A/G beads together and incubating, during which the Ruc-antigen fusion become immobilized on the A/G beads, antigen-specific antibody is quantitated by washing the beads and adding coelenterazine substrate and measuring light production. We have characterized this technology with sera from patients having three different types of cancers. We show that 20-85% of these sera contain significant titers of antibodies against at least one of five frequently mutated and/or overexpressed tumor-associated proteins. Five of six colon cancer sera tested gave responses that were statistically significantly greater than the average plus three standard deviations of 10 control sera. The results of competition experiments, preincubating positive sera with unmodified E. coli-produced antigens, varied dramatically. This technology has several advantages over current quantitative immunoassays including its relative simplicity, its avoidance of problems associated with E. coli-produced antigens and its use of antigens that can carry mammalian or disease-specific post-translational modifications. This assay should be generally useful for analyzing sera for antibodies recognizing any protein or its post-translational modifications.

A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins

PubMed Central

Burbelo, Peter D; Goldman, Radoslav; Mattson, Thomas L

2005-01-01

Background Assays detecting human antigen-specific antibodies are medically useful. However, the usefulness of existing simple immunoassay formats is limited by technical considerations such as sera antibodies to contaminants in insufficiently pure antigen, a problem likely exacerbated when antigen panels are screened to obtain clinically useful data. Results We developed a novel and simple immunoprecipitation technology for identifying clinical sera containing antigen-specific antibodies and for generating quantitative antibody response profiles. This method is based on fusing protein antigens to an enzyme reporter, Renilla luciferase (Ruc), and expressing these fusions in mammalian cells, where mammalian-specific post-translational modifications can be added. After mixing crude extracts, sera and protein A/G beads together and incubating, during which the Ruc-antigen fusion become immobilized on the A/G beads, antigen-specific antibody is quantitated by washing the beads and adding coelenterazine substrate and measuring light production. We have characterized this technology with sera from patients having three different types of cancers. We show that 20–85% of these sera contain significant titers of antibodies against at least one of five frequently mutated and/or overexpressed tumor-associated proteins. Five of six colon cancer sera tested gave responses that were statistically significantly greater than the average plus three standard deviations of 10 control sera. The results of competition experiments, preincubating positive sera with unmodified E. coli-produced antigens, varied dramatically. Conclusion This technology has several advantages over current quantitative immunoassays including its relative simplicity, its avoidance of problems associated with E. coli-produced antigens and its use of antigens that can carry mammalian or disease-specific post-translational modifications. This assay should be generally useful for analyzing sera for antibodies recognizing any protein or its post-translational modifications. PMID:16109166

Quantitative detection of crystalline lysine supplementation in poultry feeds using a rapid bacterial bioluminescence assay.

PubMed

Zabala Díaz, I B; Ricke, S C

2003-08-01

Lysine is an essential amino acid for both humans and animals; and it is usually the first or second limiting amino acid in most formulated diets. In order to estimate the lysine content in feeds and feed sources, rapid amino acid bioassays have been developed. The objective of this work is to assess a rapid assay for lysine supplementation in chicken feeds, using a luminescent Escherichia coli lysine-auxotrophic strain, to avoid prior thermal sterilization. An E. coli lysine auxotroph carrying a plasmid with lux genes was used as the test organism. The lysine assay was conducted using depleted auxotrophic cells in lysine samples. Luminescence was measured with a Dynex MLX luminometer after addition of the aldehyde substrate. Growth response (monitored as optical density at 600 nm) and light emission response of the assay E. coli strain were monitored to generate standard curves. Bioluminescent analysis of feed samples indicated that the method works well in the presence of a complex feed matrix. Comparison of both optical density and luminescent-based methods indicated that, when the assay takes place under optimal conditions, both methodologies correlated well ( r(2)=0.99). Except for the 0.64% lysine-supplemented feed, estimates for lysine based on the bacterial assay were over 80% (82-97%) of the theoretical values. Animal data showed that the bacterial bioluminescent method correlated well with the chick bioassay when diets with different levels of lysine supplementation were assayed for lysine bioavailability ( r(2)=0.97). Luminescent methodology coupled with a bacterial growth assay is a promising technique to assess lysine availability in supplemented animal feeds.

A three-domain Kazal-type serine proteinase inhibitor exhibiting domain inhibitory and bacteriostatic activities from freshwater crayfish Procambarus clarkii.

PubMed

Li, Xin-Cang; Wang, Xian-Wei; Wang, Zong-Heng; Zhao, Xiao-Fan; Wang, Jin-Xing

2009-12-01

In crustaceans, Kazal-type serine proteinase inhibitors in hemolymph are believed to function as regulators of the host-defense reactions or inhibitors against proteinases from microorganisms. In this study, we report a Kazal-type serine proteinase inhibitor, named hcPcSPI1, from freshwater crayfish (Procambarus clarkii). We found that hcPcSPI1 is composed of a putative signal peptide, an RGD motif, and three tandem Kazal-type domains with the domain P1 residues L, L and E, respectively. Mainly, hcPcSPI1 was detected in hemocytes as well as in the heart, gills, and intestine at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that hcPcSPI1 in hemocytes was upregulated by the stimulation of Esherichia coli (8099) or became decreased after a white spot syndrome virus (WSSV) challenge. In addition, hcPcSPI1 and its three independent domains were overexpressed and purified to explore their potential functions. All four proteins inhibited subtilisin A and proteinase K, but not alpha-chymotypsin or trypsin. Recombinant hcPcSPI1 could firmly attach to Gram-negative bacteria E. coli and Klebsiella pneumoniae; Gram-positive bacteria Bacillus subtilis, Bacillus thuringiensis and Staphylococcus aureus; fungi Candida albicans and Saccharomyce cerevisiae, and only domain 1 was responsible for the binding to E. coli and S. aureus. In addition, recombinant hcPcSPI1 was also found to possess bacteriostatic activity against the B. subtilis and B. thuringiensis. Domains 2 and 3 contributed mainly to these bacteriostatic activities. All results suggested that hcPcSPI1 might play important roles in the innate immunity of crayfish.

An examination of water quality indicators in swim sites located in the upper Los Angeles River Watershed

NASA Astrophysics Data System (ADS)

Lee, C. M.; Morris, K.; Fingland, N. K.; Johnstone, K.; Pendleton, L.; Ponce, A.; Tang, C.; Griffith, J. F.; Steele, N. L.

2013-12-01

Multiple sites in the upper Los Angeles River watershed were sampled during summer 2012 and measured for Escherichia coli, enterococci, and Clostridium perfringens (vegetative cells and spores) using culture-based analyses and preserved for quantitative polymerase chain reaction (qPCR) analysis. The objective of this work includes the characterization of how well indicators correlated with each other, with respect to background levels and to 'spikes' from background, possibly indicative of a pollution input, environmental/physicochemical parameters, as well as in the context of recreational water quality standards. The 2nd objective of this work was to evaluate the economic impact of implementing qPCR at our study sites for rapid water quality monitoring. None of the species of indicators correlated well with each other (R2 < 0.1) across sites and dates when the sample set was examined in its entirety, though C. perfringens vegetative cells and spores were moderately correlated (R2 = 0.31, p = 0.07). The observation of concentration 'spikes' against background levels, suggesting a potential input of contamination, were observed on holiday sampling days and will be examined further. In general, the number of swimmers present was not linked with indicator concentrations; however, incidence of water quality exceedances (for E. coli 235 CFU or MPN/100 mL sample) were more likely to occur on the weekend or holidays (for E. coli, , suggesting that the presence/absence of swimmers may be an important variable at our sites. Clostridium perfringens may be a useful indicator at our study sites, as a comparison of vegetative to endospore forms of this organism may be used to understand how recently a contamination event or input occurred.

Innate Immune Responses of Drosophila melanogaster Are Altered by Spaceflight

PubMed Central

Marcu, Oana; Lera, Matthew P.; Sanchez, Max E.; Levic, Edina; Higgins, Laura A.; Shmygelska, Alena; Fahlen, Thomas F.; Nichol, Helen; Bhattacharya, Sharmila

2011-01-01

Alterations and impairment of immune responses in humans present a health risk for space exploration missions. The molecular mechanisms underpinning innate immune defense can be confounded by the complexity of the acquired immune system of humans. Drosophila (fruit fly) innate immunity is simpler, and shares many similarities with human innate immunity at the level of molecular and genetic pathways. The goals of this study were to elucidate fundamental immune processes in Drosophila affected by spaceflight and to measure host-pathogen responses post-flight. Five containers, each containing ten female and five male fruit flies, were housed and bred on the space shuttle (average orbit altitude of 330.35 km) for 12 days and 18.5 hours. A new generation of flies was reared in microgravity. In larvae, the immune system was examined by analyzing plasmatocyte number and activity in culture. In adults, the induced immune responses were analyzed by bacterial clearance and quantitative real-time polymerase chain reaction (qPCR) of selected genes following infection with E. coli. The RNA levels of relevant immune pathway genes were determined in both larvae and adults by microarray analysis. The ability of larval plasmatocytes to phagocytose E. coli in culture was attenuated following spaceflight, and in parallel, the expression of genes involved in cell maturation was downregulated. In addition, the level of constitutive expression of pattern recognition receptors and opsonins that specifically recognize bacteria, and of lysozymes, antimicrobial peptide (AMP) pathway and immune stress genes, hallmarks of humoral immunity, were also reduced in larvae. In adults, the efficiency of bacterial clearance measured in vivo following a systemic infection with E. coli post-flight, remained robust. We show that spaceflight altered both cellular and humoral immune responses in Drosophila and that the disruption occurs at multiple interacting pathways. PMID:21264297

Quantitative microbial risk assessment (QMRA) shows increased public health risk associated with exposure to river water under conditions of riverbed sediment resuspension.

PubMed

Abia, Akebe Luther King; Ubomba-Jaswa, Eunice; Genthe, Bettina; Momba, Maggy Ndombo Benteke

2016-10-01

Although higher microbial concentrations have been reported in sediments than in the overlying water column, most quantitative microbial risk assessment (QMRA) studies have not clearly indicated the contribution of sediment-borne pathogens to estimated risks. Thus, the present study aimed at determining the public health risk associated with exposure to pathogenic bacteria in polluted river water under undisturbed conditions and conditions of sediment resuspension in the Apies River, Gauteng, South Africa. Microbial pathogens were isolated and identified using culture and molecular methods. The beta-Poisson dose-response model was used to estimate the probability of infection (Pi) with the various pathogens, following accidental/intentional ingestion of 1mL or 100mL (or 50mL) of untreated river water. Mean wet season Escherichia coli counts ranged between 5.8E+01 and 8.8E+04MPN/100mL (water column) and between 2.40E+03 and 1.28E+05MPN/100mL (sediments). Mean dry season E. coli counts ranged between 5.11E+00 and 3.40E+03MPN/100mL (water column) and between 5.09E+00 and 6.30E+03MPN/100mL (sediments). Overall (water and sediments) Vibrio cholerae was the most detected pathogen (58.8%) followed by Salmonella spp. (23.9%) and Shigella (10.1%). Ingestion of 1mL of river water could lead to 0%-4% and 1%-74% Pi with E. coli during the dry and wet season, respectively. During the dry season, the Pi with V. cholerae, Salmonella spp. and Shigella spp. were 0%-1.39%, 0%-4.11% and 0%-0.16% respectively, depending on volume of water ingested. The risks of infections with all microorganisms increased during the wet season. A 2-log increase in water E. coli count following sediments disturbance led to approximately 10 times higher Pi with E. coli than when sediments were undisturbed. Therefore, the use of the untreated water from the Apies River for drinking, household purposes or recreational activities poses a potential health risk to the users of the river. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteomic analysis reveals protein expression differences in Escherichia coli strains associated with persistent versus transient mastitis

USDA-ARS?s Scientific Manuscript database

Escherichia coli is a leading cause of bacterial mastitis in dairy cattle. Typically this infection is transient in nature, causing an infection that lasts 2-3 days. However, in a minority of cases, E. coli has been shown to cause a persistent intramammary infection. The mechanisms that allow for...

Contaminated Stream Water as Source for Escherichia coli O157 Illness in Children.

PubMed

Probert, William S; Miller, Glen M; Ledin, Katya E

2017-07-01

In May 2016, an outbreak of Shiga toxin-producing Escherichia coli O157 infections occurred among children who had played in a stream flowing through a park. Analysis of E. coli isolates from the patients, stream water, and deer and coyote scat showed that feces from deer were the most likely source of contamination.

40 CFR 142.14 - Records kept by States.

Code of Federal Regulations, 2011 CFR

2011-07-01

...)—Any decision to allow a public water system to forgo fecal coliform or E. coli testing on a total...-positive or E. coli- positive, as provided in § 141.21(e)(2). (6) Records of analysis for other than... part 141, subpart W of this chapter. (i) Results of source water E. coli and Cryptosporidium monitoring...

Prognostic importance of gram-negative intestinal colonization preceding pancreatic infection in severe acute pancreatitis. Results of a controlled clinical trial of selective decontamination.

PubMed

Luiten, E J; Hop, W C; Endtz, H P; Bruining, H A

1998-05-01

To establish, firstly, whether gram-negative (re)-colonization of the gut leads to an increased risk of gram-negative pancreatic infections and whether this event is time-related and, secondly, whether the difference in the quantity and quality of micro-organisms colonizing the digestive tract influences morbidity and mortality. Prospective analysis of the results of systematic semi-quantitative cultures of several body areas taken from patients with severe acute pancreatitis, during a controlled multicenter trial of adjuvant selective decontamination. Surgical intensive care units of 16 hospitals. A total of 2,159 semi-quantitative cultures from the oropharynx, rectum and pancreatic tissues taken from 90 patients were analyzed. Surveillance cultures from the oropharynx and rectum were taken on admission and repeated twice weekly and from the (peri)-pancreatic devitalized tissues (i. e. necrosis) at every relaparotomy and from drainage. All gram-negative pancreatic infections were preceded by intestinal colonization with the same micro-organisms. The risk of developing a pancreatic infection following gram-negative intestinal colonization (15/42 patients) was significantly higher as compared to patients without gram-negative colonization (0/10 patients) (p < 0.001) or to patients in whom E. coli was the only intestinal micro-organism cultured (0/30 patients) (p < 0.001). The occurrence of intestinal E. coli did not increase the risk of pancreatic infection. Gram-negative colonization of the rectum and oropharynx significantly correlated with the later development of pancreatic infection: relative risks 73.7 (p < 0.001) and 13.6 (p < 0.001), respectively. However, when both areas were evaluated simultaneously, the rectum was more significant (p < 0.001). The severity of intestinal intestinal colonization until the moment of pancreatic infection showed an increase in time in all 15 patients. In 11 of 15 patients (73%) these infections occurred within 1 week following the first isolation from the digestive tract. Gram-negative intestinal colonization was associated with a 3.7 fold increased mortality risk (p = 0.004). Gram-negative intestinal colonization, E. coli excepted, is an early prognostic parameter in patients in whom pancreatic infection has not yet occurred and represents a significantly increased risk of pancreatic infections and mortality.

Rapid enumeration of viable bacteria by image analysis

NASA Technical Reports Server (NTRS)

Singh, A.; Pyle, B. H.; McFeters, G. A.

1989-01-01

A direct viable counting method for enumerating viable bacteria was modified and made compatible with image analysis. A comparison was made between viable cell counts determined by the spread plate method and direct viable counts obtained using epifluorescence microscopy either manually or by automatic image analysis. Cultures of Escherichia coli, Salmonella typhimurium, Vibrio cholerae, Yersinia enterocolitica and Pseudomonas aeruginosa were incubated at 35 degrees C in a dilute nutrient medium containing nalidixic acid. Filtered samples were stained for epifluorescence microscopy and analysed manually as well as by image analysis. Cells enlarged after incubation were considered viable. The viable cell counts determined using image analysis were higher than those obtained by either the direct manual count of viable cells or spread plate methods. The volume of sample filtered or the number of cells in the original sample did not influence the efficiency of the method. However, the optimal concentration of nalidixic acid (2.5-20 micrograms ml-1) and length of incubation (4-8 h) varied with the culture tested. The results of this study showed that under optimal conditions, the modification of the direct viable count method in combination with image analysis microscopy provided an efficient and quantitative technique for counting viable bacteria in a short time.

Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells.

PubMed

Wu, Yonghong; Wu, Min; He, Guowei; Zhang, Xiao; Li, Weiguang; Gao, Yan; Li, Zhihui; Wang, Zhaoyan; Zhang, Chenggang

2012-04-01

In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Quantitative high-throughput population dynamics in continuous-culture by automated microscopy.

PubMed

Merritt, Jason; Kuehn, Seppe

2016-09-12

We present a high-throughput method to measure abundance dynamics in microbial communities sustained in continuous-culture. Our method uses custom epi-fluorescence microscopes to automatically image single cells drawn from a continuously-cultured population while precisely controlling culture conditions. For clonal populations of Escherichia coli our instrument reveals history-dependent resilience and growth rate dependent aggregation.

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

PubMed

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

2016-01-01

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

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

PubMed Central

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

2016-01-01

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

Label-free bimodal waveguide immunosensor for rapid diagnosis of bacterial infections in cirrhotic patients.

PubMed

Maldonado, Jesús; González-Guerrero, Ana Belén; Domínguez, Carlos; Lechuga, Laura M

2016-11-15

Spontaneous bacterial peritonitis is an acute bacterial infection of ascitic fluid; it has a high incidence in cirrhotic patients and it is associated with high mortality. In such a situation, early diagnosis and treatment is crucial for the survival of the patient. However, bacterial analysis in ascitic fluid is currently based on culture methods, which are time-consuming and laborious. We report here the application of a photonic interferometer biosensor based on a bimodal waveguide (BiMW) for the rapid and label-free detection of bacteria directly in ascitic fluid. The device consists of a straight waveguide in which two modes of the same polarization interfere while interacting with the external medium through their evanescent fields. A bimolecular event occurring on the sensor area of the device (e.g. capturing bacteria) will differently affect each light mode, inducing a variation in the phase of the light exiting at the output of the waveguide. In this work, we demonstrate the quantitative detection of Bacillus cereus in buffer medium and Escherichia coli in undiluted ascitic fluid from cirrhotic patients. In the case of Bacillus cereus detection, the device was able to specifically detect bacteria at relevant concentrations in 12.5min and in the case of Escherichia coli detection, the analysis time was 25min. Extrapolation of the data demonstrated that the detection limits of the biosensor could reach few bacteria per milliliter. Based on the results obtained, we consider that the BiMW biosensor is positioned as a promising new clinical tool for user-friendly, cost-effective and real-time microbiological analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Assessing host-specificity of Escherichia coli using a supervised learning logic-regression-based analysis of single nucleotide polymorphisms in intergenic regions.

PubMed

Zhi, Shuai; Li, Qiaozhi; Yasui, Yutaka; Edge, Thomas; Topp, Edward; Neumann, Norman F

2015-11-01

Host specificity in E. coli is widely debated. Herein, we used supervised learning logic-regression-based analysis of intergenic DNA sequence variability in E. coli in an attempt to identify single nucleotide polymorphism (SNP) biomarkers of E. coli that are associated with natural selection and evolution toward host specificity. Seven-hundred and eighty strains of E. coli were isolated from 15 different animal hosts. We utilized logic regression for analyzing DNA sequence data of three intergenic regions (flanked by the genes uspC-flhDC, csgBAC-csgDEFG, and asnS-ompF) to identify genetic biomarkers that could potentially discriminate E. coli based on host sources. Across 15 different animal hosts, logic regression successfully discriminated E. coli based on animal host source with relatively high specificity (i.e., among the samples of the non-target animal host, the proportion that correctly did not have the host-specific marker pattern) and sensitivity (i.e., among the samples from a given animal host, the proportion that correctly had the host-specific marker pattern), even after fivefold cross validation. Permutation tests confirmed that for most animals, host specific intergenic biomarkers identified by logic regression in E. coli were significantly associated with animal host source. The highest level of biomarker sensitivity was observed in deer isolates, with 82% of all deer E. coli isolates displaying a unique SNP pattern that was 98% specific to deer. Fifty-three percent of human isolates displayed a unique biomarker pattern that was 98% specific to humans. Twenty-nine percent of cattle isolates displayed a unique biomarker that was 97% specific to cattle. Interestingly, even within a related host group (i.e., Family: Canidae [domestic dogs and coyotes]), highly specific SNP biomarkers (98% and 99% specificity for dog and coyotes, respectively) were observed, with 21% of dog E. coli isolates displaying a unique dog biomarker and 61% of coyote isolates displaying a unique coyote biomarker. Application of a supervised learning method, such as logic regression, to DNA sequence analysis at certain intergenic regions demonstrates that some E. coli strains may evolve to become host-specific. Copyright © 2015 Elsevier Inc. All rights reserved.

Disclosing respiratory co-infections: a broad-range panel assay for avian respiratory pathogens on a nanofluidic PCR platform.

PubMed

Croville, Guillaume; Foret, Charlotte; Heuillard, Pauline; Senet, Alexis; Delpont, Mattias; Mouahid, Mohammed; Ducatez, Mariette F; Kichou, Faouzi; Guerin, Jean-Luc

2018-06-01

Respiratory syndromes (RS) are among the most significant pathological conditions in edible birds and are caused by complex coactions of pathogens and environmental factors. In poultry, low pathogenic avian influenza A viruses, metapneumoviruses, infectious bronchitis virus, infectious laryngotracheitis virus, Mycoplasma spp. Escherichia coli and/or Ornithobacterium rhinotracheale in turkeys are considered as key co-infectious agents of RS. Aspergillus sp., Pasteurella multocida, Avibacterium paragallinarum or Chlamydia psittaci may also be involved in respiratory outbreaks. An innovative quantitative PCR method, based on a nanofluidic technology, has the ability to screen up to 96 samples with 96 pathogen-specific PCR primers, at the same time, in one run of real-time quantitative PCR. This platform was used for the screening of avian respiratory pathogens: 15 respiratory agents, including viruses, bacteria and fungi potentially associated with respiratory infections of poultry, were targeted. Primers were designed and validated for SYBR green real-time quantitative PCR and subsequently validated on the Biomark high throughput PCR nanofluidic platform (Fluidigm©, San Francisco, CA, USA). As a clinical assessment, tracheal swabs were sampled from turkeys showing RS and submitted to this panel assay. Beside systematic detection of E. coli, avian metapneumovirus, Mycoplasma gallisepticum and Mycoplasma synoviae were frequently detected, with distinctive co-infection patterns between French and Moroccan flocks. This proof-of-concept study illustrates the potential of such panel assays for unveiling respiratory co-infection profiles in poultry.

Multiple-locus variable number of tandem repeat analysis (MLVA) of Irish verocytotoxigenic Escherichia coli O157 from feedlot cattle: uncovering strain dissemination routes.

PubMed

Murphy, Mary; Minihan, Donal; Buckley, James F; O'Mahony, Micheál; Whyte, Paul; Fanning, Séamus

2008-01-24

The identification of the routes of dissemination of Escherichia coli (E. coli) O157 through a cohort of cattle is a critical step to control this pathogen at farm level. The aim of this study was to identify potential routes of dissemination of E. coli O157 using Multiple-Locus Variable number of tandem repeat Analysis (MLVA). Thirty-eight environmental and sixteen cattle faecal isolates, which were detected in four adjacent pens over a four-month period were sub-typed. MLVA could separate these isolates into broadly defined clusters consisting of twelve MLVA types. Strain diversity was observed within pens, individual cattle and the environment. Application of MLVA is a broadly useful and convenient tool when applied to uncover the dissemination of E. coli O157 in the environment and in supporting improved on-farm management of this important pathogen. These data identified diverse strain types based on amplification of VNTR markers in each case.

Identification and characterization of Escherichia coli RS218-derived islands in the pathogenesis of E. coli meningitis.

PubMed

Xie, Yi; Kolisnychenko, Vitaliy; Paul-Satyaseela, Maneesh; Elliott, Simon; Parthasarathy, Geetha; Yao, Yufeng; Plunkett, Guy; Blattner, Frederick R; Kim, Kwang Sik

2006-08-01

Escherichia coli K1 is the most common gram-negative bacterium causing neonatal meningitis, but the mechanisms by which E. coli K1 causes meningitis are not clear. We identified 22 E. coli RS218-derived genomic islands (RDIs), using a comparative genome analysis of meningitis-causing E. coli K1 strain RS218 (O18:K1:H7) and laboratory K-12 strain MG1655. Series of RDI deletion mutants were constructed and examined for phenotypes relevant to E. coli K1 meningitis. We identified 9 RDI deletion mutants (RDI 1, 4, 7, 12, 13, 16, 20, 21, and 22) that exhibited defects in meningitis development. RDI 16 and 21 mutants had profound defects in the induction of a high level of bacteremia in neonatal rats, and RDI 4 mutants exhibited a moderate defect in the induction of bacteremia. RDI 1 and 22 mutants showed defects in the ability to invade human brain microvascular endothelial cells (HBMECs), and RDI 12 mutants were defective in the ability to bind to HBMECs. RDI 13 and 20 mutants were defective in the ability to both bind to and invade HBMECs. RDI 7 mutants were defective in the induction of bacteremia and in the ability to both bind to and invade HBMECs. These results provide a framework for the future discovery and analysis of bacteremia and meningitis caused by E. coli K1 strain RS218.

Multiple-locus variable-nucleotide tandem repeat subtype analysis implicates European starlings as biological vectors for Escherichia coli O157:H7 in Ohio, USA.

PubMed

Williams, M L; Pearl, D L; Lejeune, J T

2011-10-01

To provide molecular epidemiological evidence of avian transmission of Escherichia coli O157:H7 between dairy farms in Ohio, this study was designed to identify genetic relatedness between isolates originating from bovine faecal samples and intestinal contents of European starlings captured on these farms. During a three-year period (2007-2009), cattle (n = 9000) and starlings (n = 430) on 150 different dairy farms in northern Ohio were sampled for the presence of E. coli O157:H7. Isolates were subjected to multiple-locus variable-nucleotide tandem repeat analysis (MLVA). Distinct allelic groups were identified on most farms; however, isolates clustering into three MLVA groups originated from both cattle and birds on different farms. Sharing of indistinguishable epidemiologically linked E. coli O157 MLVA subtypes between starlings and cattle on different farms supports the hypothesis that these birds contribute to the transmission of E. coli O157:H7 between dairy farms. A continued need exists to identify and to improve preharvest measures for controlling E. coli O157:H7. Controlling wildlife intrusion, particularly European starlings, on livestock operations, may be an important strategy for reducing dissemination of E. coli O157:H7 between farms and thereby potentially decreasing the on-farm prevalence of E. coli O157:H7 and enhancing the safety of the food supply. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Inhibition of verocytotoxigenic Escherichia coli by antimicrobial peptides caseicin A and B and the factors affecting their antimicrobial activities.

PubMed

McDonnell, Mary J; Rivas, Lucia; Burgess, Catherine M; Fanning, Séamus; Duffy, Geraldine

2012-02-15

The antimic robial activities of caseicin A and B antimicrobial peptides (AMPs) were assessed against a selection of verocytotoxigenic Escherichia coli (VTEC) strains (n=11), other bacterial pathogenic and spoilage bacteria (n=7), using a model broth system. The ability of the AMPs to retain their antimicrobial activities against a strain of E. coli O157:H7 380-94 under various test conditions (pH, temperature, water activity, sodium chloride concentrations, inoculum size and the presence of competitive microflora) was assessed and the minimum inhibitory concentrations (MIC) and number of surviving E. coli O157:H7 calculated. The mean number of VTEC surviving after exposure to 2 mg/ml caseicin A and B was reduced by 4.96 and 4.19 log(10) cfu/ml compared to the respective controls. The susceptibility of E. coli O157:H7 to the caseicin AMPs decreased as temperature, pH, water activity and inoculum size were reduced. The presence of sodium chloride (0.5-2.5%) did not affect the activity of caseicin A (p>0.05), however it did inhibit the activity of caseicin B. The presence of a competitive microflora cocktail did not significantly (p>0.05) affect the activities of the AMPs for the majority of the concentrations tested. Using a quantitative PCR assay, the levels of verotoxins (vt1 and vt2) expressed by E. coli O157:H7 following exposure to a sub-inhibitory concentration (0.5 mg/ml) of caseicin A showed that the verotoxin levels did not differ from the levels produced by the control cultures. The antimicrobial activity of caseicin A against E. coli O157:H7 was also tested in a model rumen system, however concentrations of ≥2 mg/ml did not significantly (p>0.05) reduce E. coli O157:H7 numbers in the model system over a 24 h period. The application of caseicin AMPs in food and/or animal production may be valuable in combination with other antimicrobials although further research is required. Copyright © 2011 Elsevier B.V. All rights reserved.

Quantitative assessment of thermodynamic constraints on the solution space of genome-scale metabolic models.

PubMed

Hamilton, Joshua J; Dwivedi, Vivek; Reed, Jennifer L

2013-07-16

Constraint-based methods provide powerful computational techniques to allow understanding and prediction of cellular behavior. These methods rely on physiochemical constraints to eliminate infeasible behaviors from the space of available behaviors. One such constraint is thermodynamic feasibility, the requirement that intracellular flux distributions obey the laws of thermodynamics. The past decade has seen several constraint-based methods that interpret this constraint in different ways, including those that are limited to small networks, rely on predefined reaction directions, and/or neglect the relationship between reaction free energies and metabolite concentrations. In this work, we utilize one such approach, thermodynamics-based metabolic flux analysis (TMFA), to make genome-scale, quantitative predictions about metabolite concentrations and reaction free energies in the absence of prior knowledge of reaction directions, while accounting for uncertainties in thermodynamic estimates. We applied TMFA to a genome-scale network reconstruction of Escherichia coli and examined the effect of thermodynamic constraints on the flux space. We also assessed the predictive performance of TMFA against gene essentiality and quantitative metabolomics data, under both aerobic and anaerobic, and optimal and suboptimal growth conditions. Based on these results, we propose that TMFA is a useful tool for validating phenotypes and generating hypotheses, and that additional types of data and constraints can improve predictions of metabolite concentrations. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Effects of in-feed Chlortetracycline Prophylaxis of beef cattle on animal health and antimicrobial-resistant Escherichia coli

USDA-ARS?s Scientific Manuscript database

Objective: The objective of this study was to evaluate the effect of a one-time, five-day in-feed CTC prophylaxis on animal health (morbidity and body weight gain), occurrence of TETr E. coli, and occurrence of 3GCr E. coli over a four-month follow-up period. Experimental Design & Analysis: We eval...

Identification and pathogenomic analysis of an Escherichia coli strain producing a novel Shiga toxin 2 subtype

USDA-ARS?s Scientific Manuscript database

Shiga toxin (Stx) is the key virulent factor in Shiga toxin-producing Escherichia coli (STEC). To date, three Stx1 subtypes and Seven Stx2 subtypes have been described in E. coli, which were found to differ in receptor preference and toxin potency. Here, we identified a novel Stx2 subtype designated...

Escherichia coli bacteria density in relation to turbidity, streamflow characteristics, and season in the Chattahoochee River near Atlanta, Georgia, October 2000 through September 2008—Description, statistical analysis, and predictive modeling

USGS Publications Warehouse

Lawrence, Stephen J.

2012-01-01

Regression analyses show that E. coli density in samples was strongly related to turbidity, streamflow characteristics, and season at both sites. The regression equation chosen for the Norcross data showed that 78 percent of the variability in E. coli density (in log base 10 units) was explained by the variability in turbidity values (in log base 10 units), streamflow event (dry-weather flow or stormflow), season (cool or warm), and an interaction term that is the cross product of streamflow event and turbidity. The regression equation chosen for the Atlanta data showed that 76 percent of the variability in E. coli density (in log base 10 units) was explained by the variability in turbidity values (in log base 10 units), water temperature, streamflow event, and an interaction term that is the cross product of streamflow event and turbidity. Residual analysis and model confirmation using new data indicated the regression equations selected at both sites predicted E. coli density within the 90 percent prediction intervals of the equations and could be used to predict E. coli density in real time at both sites.

Detection of human-derived fecal contamination in Puerto Rico using carbamazepine, HF183 Bacteroides, and fecal indicator bacteria.

PubMed

Wade, Christina; Otero, Ernesto; Poon-Kwong, Brennan; Rozier, Ralph; Bachoon, Dave

2015-12-30

The level of fecal pollution in 17 sites in Puerto Rico was determined by Escherichia coli (E.coli) enumeration using an enzyme substrate medium and Quanti-Tray®/2000. Human fecal pollution was identified using an enzyme-linked immunosorbent assay for the detection of carbamazepine (CBZ) and quantitative polymerase chain reaction (qPCR) detection of the human Bacteroides marker, HF183. Carbamazepine was detected in 16 out of 17 sites, including Condado Lagoon, a popular recreational area. Elevated E.coli levels (>410 CFU 100 mL(-1)) were detected in 13 sites. Average CBZ concentrations ranged from 0.005 μg L(-1) to 0.482 μg L(-1) and 7 sites were positive for HF183. Higher CBZ concentrations were associated with the detection of HF183 (Mann-Whitney test; U=42.0; df=7; 1-tailed P value=0.013). This was the second study to determine surface water concentrations of CBZ in the Caribbean and the first in Puerto Rico. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of the putative neutrophil-generated toxin, hypochlorous acid, on membrane permeability and transport systems of Escherichia coli.

PubMed Central

Albrich, J M; Gilbaugh, J H; Callahan, K B; Hurst, J K

1986-01-01

Titrimetric addition of hypochlorous acid (HOCl) or chloramine (NH2Cl) to suspensions of Escherichia coli decreases their ability to accumulate 14C-labeled glutamine, proline, thiomethylgalactoside, and leucine in a manner that approximately coincides with loss of cell viability; quantitative differences in cellular response are observed with the two oxidants. Inhibition of beta-galactosidase activity in E. coli ML-35, a strain lacking functional lactose permease, is complex and also depends upon the identity of the oxidant. Membrane proton conductivities and glycerol permeabilities are unchanged by addition of HOCl or NH2Cl in excess of that required for inactivation. The combined results are interpreted to indicate that the locus of HOCl attack is the cell envelope, that HOCl inactivation does not occur by loss of membrane structural integrity, that loss of transport function can be identified with either selective oxidative inhibition of the transport proteins or loss of cellular metabolic energy, and that different mechanisms of inactivation may exist for HOCl and NH2Cl. PMID:3013936

Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System.

PubMed

Marshall, Ryan; Maxwell, Colin S; Collins, Scott P; Jacobsen, Thomas; Luo, Michelle L; Begemann, Matthew B; Gray, Benjamin N; January, Emma; Singer, Anna; He, Yonghua; Beisel, Chase L; Noireaux, Vincent

2018-01-04

CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression-all without protein purification or live cells. We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses. Copyright © 2017 Elsevier Inc. All rights reserved.

Construction of a novel shuttle vector for use in Gluconobacter oxydans.

PubMed

Zhang, Lin; Lin, Jinping; Ma, Yushu; Wei, Dongzhi; Sun, Ming

2010-11-01

A shuttle vector pZL1 which can replicate both in Gluconobacter oxydans and Escherichia coli was constructed based on G. oxydans DSM2003 cryptic plasmid pGOX3, a homology of G. oxydans 621H pGOX3, and E. coli cloning vector pUC18. It was found to be stably maintained in G. oxydans during the serial subcultures in the absence of antibiotic pressure for 144 h. With pGOX3 as the reference sample, the relative copy number of pZL1 in G. oxydans is 13 determined by real-time fluorescence quantitative PCR (qPCR). The copy number of pZL1 is much higher than pBBR1MCS5 in E. coli. The vector pZL1 contains six commonly used restriction endonuclease sites, HindIII, SalI, XbaI, BamHI, SmaI, KpnI, and SacI, and is easy to manipulate in molecular biology experiments. The shuttle vector was used to express a reporter protein wasabi successfully in G. oxydans DSM2003 under the control of the tufB promoter.

H2S as an Indicator of Water Supply Vulnerability and Health Risk in Low-Resource Settings: A Prospective Cohort Study

PubMed Central

Khush, Ranjiv S.; Arnold, Benjamin F.; Srikanth, Padma; Sudharsanam, Suchithra; Ramaswamy, Padmavathi; Durairaj, Natesan; London, Alicia G.; Ramaprabha, Prabhakar; Rajkumar, Paramasivan; Balakrishnan, Kalpana; Colford Jr., John M.

2013-01-01

In this large-scale longitudinal study conducted in rural Southern India, we compared a presence/absence hydrogen sulfide (H2S) test with quantitative assays for total coliforms and Escherichia coli as measures of water quality, health risk, and water supply vulnerability to microbial contamination. None of the three indicators showed a significant association with child diarrhea. The presence of H2S in a water sample was associated with higher levels of total coliform species that may have included E. coli but that were not restricted to E. coli. In addition, we observed a strong relationship between the percent positive H2S test results and total coliform levels among water source samples (R2 = 0.87). The consistent relationships between H2S and total coliform levels indicate that presence/absence of H2S tests provide a cost-effective option for assessing both the vulnerability of water supplies to microbial contamination and the results of water quality management and risk mitigation efforts. PMID:23716404

Overexpression of SOS genes in ciprofloxacin resistant Escherichia coli mutants.

PubMed

Pourahmad Jaktaji, Razieh; Pasand, Shirin

2016-01-15

Fluoroquinolones are important antibiotics for the treatment of urinary tract infections caused by Escherichia coli. Mutational studies have shown that ciprofloxacin, a member of fluoroquinolones induces SOS response and mutagenesis in pathogenic bacteria which in turn develop antibiotic resistance. However, inhibition of SOS response can increase recombination activity which in turn leads to genetic variation. The aim of this study was to measure 5 SOS genes expressions in nine E. coli mutants with different MICs for ciprofloxacin following exposure to ciprofloxacin. Gene expression was assessed by quantitative real time PCR. Gene alteration assessment was conducted by PCR amplification and DNA sequencing. Results showed that the expression of recA was increased in 5 mutants. This overexpression is not related to gene alteration, and enhances the expression of polB and umuCD genes encoding nonmutagenic and mutagenic polymerases, respectively. The direct relationship between the level of SOS expression and the level of resistance to ciprofloxacin was also indicated. It was concluded that novel therapeutic strategy that inhibits RecA activity would enhance the efficiency of common antibiotics against pathogenic bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

Need-based activation of ammonium uptake in Escherichia coli

PubMed Central

Kim, Minsu; Zhang, Zhongge; Okano, Hiroyuki; Yan, Dalai; Groisman, Alexander; Hwa, Terence

2012-01-01

The efficient sequestration of nutrients is vital for the growth and survival of microorganisms. Some nutrients, such as CO2 and NH3, are readily diffusible across the cell membrane. The large membrane permeability of these nutrients obviates the need of transporters when the ambient level is high. When the ambient level is low, however, maintaining a high intracellular nutrient level against passive back diffusion is both challenging and costly. Here, we study the delicate management of ammonium (NH4+/NH3) sequestration by E. coli cells using microfluidic chemostats. We find that as the ambient ammonium concentration is reduced, E. coli cells first maximize their ability to assimilate the gaseous NH3 diffusing into the cytoplasm and then abruptly activate ammonium transport. The onset of transport varies under different growth conditions, but always occurring just as needed to maintain growth. Quantitative modeling of known interactions reveals an integral feedback mechanism by which this need-based uptake strategy is implemented. This novel strategy ensures that the expensive cost of upholding the internal ammonium concentration against back diffusion is kept at a minimum. PMID:23010999

Escherichia coli identification and strain discrimination using nanosecond laser-induced breakdown spectroscopy

NASA Astrophysics Data System (ADS)

Diedrich, Jonathan; Rehse, Steven J.; Palchaudhuri, Sunil

2007-04-01

Three strains of Escherichia coli, one strain of environmental mold, and one strain of Candida albicans yeast have been analyzed by laser-induced breakdown spectroscopy using nanosecond laser pulses. All microorganisms were analyzed while still alive and with no sample preparation. Nineteen atomic and ionic emission lines have been identified in the spectrum, which is dominated by calcium, magnesium, and sodium. A discriminant function analysis has been used to discriminate between the biotypes and E. coli strains. This analysis showed efficient discrimination between laser-induced breakdown spectroscopy spectra from different strains of a single bacteria species.

Evaluation of Two Multiplex PCR-High-Resolution Melt Curve Analysis Methods for Differentiation of Campylobacter jejuni and Campylobacter coli Intraspecies.

PubMed

Banowary, Banya; Dang, Van Tuan; Sarker, Subir; Connolly, Joanne H; Chenu, Jeremy; Groves, Peter; Raidal, Shane; Ghorashi, Seyed Ali

2018-03-01

Campylobacter infection is a common cause of bacterial gastroenteritis in humans and remains a significant global public health issue. The capability of two multiplex PCR (mPCR)-high-resolution melt (HRM) curve analysis methods (i.e., mPCR1-HRM and mPCR2-HRM) to detect and differentiate 24 poultry isolates and three reference strains of Campylobacter jejuni and Campylobacter coli was investigated. Campylobacter jejuni and C. coli were successfully differentiated in both assays, but the differentiation power of mPCR2-HRM targeting the cadF gene was found superior to that of mPCR1-HRM targeting the gpsA gene or a hypothetical protein gene. However, higher intraspecies variation within C. coli and C. jejuni isolates was detected in mPCR1-HRM when compared with mPCR2-HRM. Both assays were rapid and required minimum interpretation skills for discrimination between and within Campylobacter species when using HRM curve analysis software.

Survival, Biofilm Formation, and Growth Potential of Environmental and Enteric Escherichia coli Strains in Drinking Water Microcosms

PubMed Central

Abberton, Cathy L.; Bereschenko, Ludmila; van der Wielen, Paul W. J. J.

2016-01-01

ABSTRACT Escherichia coli is the most commonly used indicator for fecal contamination in drinking water distribution systems (WDS). The assumption is that E. coli bacteria are of enteric origin and cannot persist for long outside their host and therefore act as indicators of recent contamination events. This study investigates the fate of E. coli in drinking water, specifically addressing survival, biofilm formation under shear stress, and regrowth in a series of laboratory-controlled experiments. We show the extended persistence of three E. coli strains (two enteric isolates and one soil isolate) in sterile and nonsterile drinking water microcosms at 8 and 17°C, with T90 (time taken for a reduction in cell number of 1 log10 unit) values ranging from 17.4 ± 1.8 to 149 ± 67.7 days, using standard plate counts and a series of (reverse transcription-)quantitative PCR [(RT-)Q-PCR] assays targeting 16S rRNA, tuf, uidA, and rodA genes and transcripts. Furthermore, each strain was capable of attaching to a surface and replicating to form biofilm in the presence of nutrients under a range of shear stress values (0.6, 2.0, and 4.4 dynes [dyn] cm−2; BioFlux system; Fluxion); however, cell numbers did not increase when drinking water flowed over the biofilm (P > 0.05 by t test). Finally, E. coli regrowth within drinking water microcosms containing polyethylene PE-100 pipe wall material was not observed in the biofilm or water phase using a combination of culturing and Q-PCR methods for E. coli. The results of this work highlight that when E. coli enters drinking water it has the potential to survive and attach to surfaces but that regrowth within drinking water or biofilm is unlikely. IMPORTANCE The provision of clean, safe drinking water is fundamental to society. WDS deliver water to consumers via a vast network of pipes. E. coli is used as an indicator organism for recent contamination events based on the premise that it cannot survive for long outside its host. A key public health concern therefore arises around the fate of E. coli on entering a WDS; its survival, ability to form a biofilm, and potential for regrowth. In particular, if E. coli bacteria have the ability to incorporate and regrow within the pipe wall biofilm of a WDS, they could reinoculate the water at a later stage. This study sheds light on the fate of environmental and enteric strains of E. coli in drinking water showing extended survival, the potential for biofilm formation under shear stress, and importantly, that regrowth in the presence of an indigenous microbial community is unlikely. PMID:27342552

Survival, Biofilm Formation, and Growth Potential of Environmental and Enteric Escherichia coli Strains in Drinking Water Microcosms.

PubMed

Abberton, Cathy L; Bereschenko, Ludmila; van der Wielen, Paul W J J; Smith, Cindy J

2016-09-01

Escherichia coli is the most commonly used indicator for fecal contamination in drinking water distribution systems (WDS). The assumption is that E. coli bacteria are of enteric origin and cannot persist for long outside their host and therefore act as indicators of recent contamination events. This study investigates the fate of E. coli in drinking water, specifically addressing survival, biofilm formation under shear stress, and regrowth in a series of laboratory-controlled experiments. We show the extended persistence of three E. coli strains (two enteric isolates and one soil isolate) in sterile and nonsterile drinking water microcosms at 8 and 17°C, with T90 (time taken for a reduction in cell number of 1 log10 unit) values ranging from 17.4 ± 1.8 to 149 ± 67.7 days, using standard plate counts and a series of (reverse transcription-)quantitative PCR [(RT-)Q-PCR] assays targeting 16S rRNA, tuf, uidA, and rodA genes and transcripts. Furthermore, each strain was capable of attaching to a surface and replicating to form biofilm in the presence of nutrients under a range of shear stress values (0.6, 2.0, and 4.4 dynes [dyn] cm(-2); BioFlux system; Fluxion); however, cell numbers did not increase when drinking water flowed over the biofilm (P > 0.05 by t test). Finally, E. coli regrowth within drinking water microcosms containing polyethylene PE-100 pipe wall material was not observed in the biofilm or water phase using a combination of culturing and Q-PCR methods for E. coli The results of this work highlight that when E. coli enters drinking water it has the potential to survive and attach to surfaces but that regrowth within drinking water or biofilm is unlikely. The provision of clean, safe drinking water is fundamental to society. WDS deliver water to consumers via a vast network of pipes. E. coli is used as an indicator organism for recent contamination events based on the premise that it cannot survive for long outside its host. A key public health concern therefore arises around the fate of E. coli on entering a WDS; its survival, ability to form a biofilm, and potential for regrowth. In particular, if E. coli bacteria have the ability to incorporate and regrow within the pipe wall biofilm of a WDS, they could reinoculate the water at a later stage. This study sheds light on the fate of environmental and enteric strains of E. coli in drinking water showing extended survival, the potential for biofilm formation under shear stress, and importantly, that regrowth in the presence of an indigenous microbial community is unlikely. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Quantitative structure-activity relationships by neural networks and inductive logic programming. I. The inhibition of dihydrofolate reductase by pyrimidines

NASA Astrophysics Data System (ADS)

Hirst, Jonathan D.; King, Ross D.; Sternberg, Michael J. E.

1994-08-01

Neural networks and inductive logic programming (ILP) have been compared to linear regression for modelling the QSAR of the inhibition of E. coli dihydrofolate reductase (DHFR) by 2,4-diamino-5-(substitured benzyl)pyrimidines, and, in the subsequent paper [Hirst, J.D., King, R.D. and Sternberg, M.J.E., J. Comput.-Aided Mol. Design, 8 (1994) 421], the inhibition of rodent DHFR by 2,4-diamino-6,6-dimethyl-5-phenyl-dihydrotriazines. Cross-validation trials provide a statistically rigorous assessment of the predictive capabilities of the methods, with training and testing data selected randomly and all the methods developed using identical training data. For the ILP analysis, molecules are represented by attributes other than Hansch parameters. Neural networks and ILP perform better than linear regression using the attribute representation, but the difference is not statistically significant. The major benefit from the ILP analysis is the formulation of understandable rules relating the activity of the inhibitors to their chemical structure.

Escherichia coli O157:H7 in Ecuador: animal reservoirs, yet no human disease.

PubMed

Trueba, Gabriel; Garcés, Verónica; V, Verónica Barragan; Colman, Rebecca E; Seymour, Meagan; Vogler, Amy J; Keim, Paul

2013-05-01

Escherichia coli O157:H7 is frequently isolated from cases of diarrhea in many industrialized countries; however, it is seldom found in developing countries. The present manuscript reports the presence of E. coli O157:H7 in Ecuadorian livestock, a country where enterohemorrhagic E. coli disease in humans has never been reported. The Ecuadorian isolates were genetically related to some strains linked to clinical cases in the United States as assessed by multiple-locus variable number tandem repeat (VNTR) analysis.

Analysis of the Genome Structure of the Nonpathogenic Probiotic Escherichia coli Strain Nissle 1917

PubMed Central

Grozdanov, Lubomir; Raasch, Carsten; Schulze, Jürgen; Sonnenborn, Ulrich; Gottschalk, Gerhard; Hacker, Jörg; Dobrindt, Ulrich

2004-01-01

Nonpathogenic Escherichia coli strain Nissle 1917 (O6:K5:H1) is used as a probiotic agent in medicine, mainly for the treatment of various gastroenterological diseases. To gain insight on the genetic level into its properties of colonization and commensalism, this strain's genome structure has been analyzed by three approaches: (i) sequence context screening of tRNA genes as a potential indication of chromosomal integration of horizontally acquired DNA, (ii) sequence analysis of 280 kb of genomic islands (GEIs) coding for important fitness factors, and (iii) comparison of Nissle 1917 genome content with that of other E. coli strains by DNA-DNA hybridization. PCR-based screening of 324 nonpathogenic and pathogenic E. coli isolates of different origins revealed that some chromosomal regions are frequently detectable in nonpathogenic E. coli and also among extraintestinal and intestinal pathogenic strains. Many known fitness factor determinants of strain Nissle 1917 are localized on four GEIs which have been partially sequenced and analyzed. Comparison of these data with the available knowledge of the genome structure of E. coli K-12 strain MG1655 and of uropathogenic E. coli O6 strains CFT073 and 536 revealed structural similarities on the genomic level, especially between the E. coli O6 strains. The lack of defined virulence factors (i.e., alpha-hemolysin, P-fimbrial adhesins, and the semirough lipopolysaccharide phenotype) combined with the expression of fitness factors such as microcins, different iron uptake systems, adhesins, and proteases, which may support its survival and successful colonization of the human gut, most likely contributes to the probiotic character of E. coli strain Nissle 1917. PMID:15292145

The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli

PubMed Central

2011-01-01

Background Escherichia coli is a model prokaryote, an important pathogen, and a key organism for industrial biotechnology. E. coli W (ATCC 9637), one of four strains designated as safe for laboratory purposes, has not been sequenced. E. coli W is a fast-growing strain and is the only safe strain that can utilize sucrose as a carbon source. Lifecycle analysis has demonstrated that sucrose from sugarcane is a preferred carbon source for industrial bioprocesses. Results We have sequenced and annotated the genome of E. coli W. The chromosome is 4,900,968 bp and encodes 4,764 ORFs. Two plasmids, pRK1 (102,536 bp) and pRK2 (5,360 bp), are also present. W has unique features relative to other sequenced laboratory strains (K-12, B and Crooks): it has a larger genome and belongs to phylogroup B1 rather than A. W also grows on a much broader range of carbon sources than does K-12. A genome-scale reconstruction was developed and validated in order to interrogate metabolic properties. Conclusions The genome of W is more similar to commensal and pathogenic B1 strains than phylogroup A strains, and therefore has greater utility for comparative analyses with these strains. W should therefore be the strain of choice, or 'type strain' for group B1 comparative analyses. The genome annotation and tools created here are expected to allow further utilization and development of E. coli W as an industrial organism for sucrose-based bioprocesses. Refinements in our E. coli metabolic reconstruction allow it to more accurately define E. coli metabolism relative to previous models. PMID:21208457

Longitudinal prevalence and molecular typing of Escherichia coli O157:H7 by use of multiple-locus variable-number tandem-repeat analysis and pulsed-field gel electrophoresis in fecal samples collected from a range-based herd of beef cattle in California.

PubMed

Kondo, Sonoko; Hoar, Bruce R; Villanueva, Veronica; Mandrell, Robert E; Atwill, Edward R

2010-11-01

To evaluate seasonal patterns and risk factors for Escherichia coli O157:H7 in feces in a beef cattle herd and determine strain diversity and transition in E coli over time by use of multiple-locus variable-number tandem-repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). 456 samples of freshly passed feces collected over a 1-year period from cattle in a range-based cow-calf operation located in the foothills of the Sierra Nevada Mountains in California. E coli O157:H7 was recovered from feces by use of immunomagnetic separation and 2 selective media. Virulence factors were detected via reverse transcriptase-PCR assay. Escherichia coli O157:H7 isolates were subtyped with MLVA and PFGE. Prevalence estimates were calculated and significant risk factors determined. A dendrogram was constructed on the basis of results of MLVA typing. Overall prevalence estimate for E coli O157:H7 was 10.5%, with the prevalence lowest during the winter. Mean temperature during the 30 days before collection of samples was significantly associated with prevalence of E coli O157:H7 in feces. Nineteen MLVA and 12 PFGE types were identified. A seasonal pattern was detected for prevalence of E coli O157:H7 in feces collected from beef cattle in California. Subtyping via MLVA and PFGE revealed a diversity of E coli O157:H7 strains in a cow-calf operation and noteworthy turnover of predominant types. Given the importance of accurately determining sources of contamination in investigations of disease outbreaks in humans, MLVA combined with PFGE should be powerful tools for epidemiologists.

Antibacterial activity and mechanism of action of ε-poly-L-lysine

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

Ye, Ruosong; Xu, Hengyi; Wan, Cuixiang

Highlights: •Antibacterial activity and mechanism of ε-PL against E. coli O157:H7 was investigated. •Critical inhibitory factors toward the growth of E. coli O157:H7 by ε-PL was analyzed. •Cell membrane integrity and cell morphology of E. coli O157:H7 was affected by ε-PL. •A positive correlation between reactive oxygen species levels and ε-PL concentration in E. coli O157:H7 cells. •ε-PL induced the expression of different genes related to oxidative/redox stress, SOS response, virulence. -- Abstract: ε-Poly-L-lysine (ε-PL) is widely used as an antibacterial agent because of its broad antimicrobial spectrum. However, the mechanism of ε-PL against pathogens at the molecular level hasmore » not been elucidated. This study investigated the antibacterial activity and mechanism of ε-PL against Escherichia coli O157:H7 CMCC44828. Propidium monoazide-PCR test results indicated that the threshold condition of ε-PL for complete membrane lysis of E. coli O157:H7 was 10 μg/mL (90% mortality for 5 μg/mL). Further verification of the destructive effect of ε-PL on cell structure was performed by atomic force microscopy and transmission electron microscopy. Results showed a positive correlation between reactive oxygen species (ROS) levels and ε-PL concentration in E. coli O157:H7 cells. Moreover, the mortality of E. coli O157:H7 was reduced when antioxidant N-acetylcysteine was added. Results from real-time quantitative PCR (RT-qPCR) indicated that the expression levels of oxidative stress genes sodA and oxyR were up-regulated 4- and 16-fold, respectively, whereas virulence genes eaeA and espA were down-regulated after ε-PL treatment. Expression of DNA damage response (SOS response) regulon genes recA and lexA were also affected by ε-PL. In conclusion, the antibacterial mechanism of ε-PL against E. coli O157:H7 may be attributed to disturbance on membrane integrity, oxidative stress by ROS, and effects on various gene expressions, such as regulation of oxidative stress, SOS response, and changes in virulence.« less

Protein disorder is positively correlated with gene expression in E. coli

PubMed Central

Paliy, Oleg; Gargac, Shawn M.; Cheng, Yugong; Uversky, Vladimir N.; Dunker, A. Keith

2009-01-01

We considered on a global scale the relationship between the predicted fraction of protein disorder and RNA and protein expression in E. coli. Fraction of protein disorder correlated positively with both measured RNA expression levels of E. coli genes in three different growth media and with predicted abundance levels of E. coli proteins. Though weak, the correlation was highly significant. Correlation of protein disorder with RNA expression did not depend on the growth rate of E. coli cultures and was not caused by a small subset of genes showing exceptionally high concordance in their disorder and expression levels. Global analysis was complemented by detailed consideration of several groups of proteins. PMID:18465893

Expression and functional analysis of the lysine decarboxylase and copper amine oxidase genes from the endophytic fungus Colletotrichum gloeosporioides ES026.

PubMed

Zhang, Xiangmei; Wang, Zhangqian; Jan, Saad; Yang, Qian; Wang, Mo

2017-06-05

Huperzine A (HupA) isolated from Huperzia serrata is an important compound used to treat Alzheimer's disease (AD). Recently, HupA was reported in various endophytic fungi, with Colletotrichum gloeosporioides ES026 previously isolated from H. serrata shown to produce HupA. In this study, we performed next-generation sequencing and de novo RNA sequencing of C. gloeosporioides ES026 to elucidate the molecular functions, biological processes, and biochemical pathways of these unique sequences. Gene ontology and Kyoto Encyclopedia of Genes and Genomes assignments allowed annotation of lysine decarboxylase (LDC) and copper amine oxidase (CAO) for their conversion of L-lysine to 5-aminopentanal during HupA biosynthesis. Additionally, we constructed a stable, high-yielding HupA-expression system resulting from the overexpression of CgLDC and CgCAO from the HupA-producing endophytic fungus C. gloeosporioides ES026 in Escherichia coli. Quantitative reverse transcription polymerase chain reaction analysis confirmed CgLDC and CgCAO expression, and quantitative determination of HupA levels was assessed by liquid chromatography high-resolution mass spectrometry, which revealed that elevated expression of CgLDC and CgCAO produced higher yields of HupA than those derived from C. gloeosporioides ES026. These results revealed CgLDC and CgCAO involvement in HupA biosynthesis and their key role in regulating HupA content in C. gloeosporioides ES026.

Evaluation of Fourier transform infrared (FT-IR) spectroscopy and chemometrics as a rapid approach for sub-typing Escherichia coli O157:H7 isolates.

PubMed

Davis, R; Paoli, G; Mauer, L J

2012-09-01

The importance of tracking outbreaks of foodborne illness and the emergence of new virulent subtypes of foodborne pathogens have created the need for rapid and reliable sub-typing methods for Escherichia coli O157:H7. Fourier transform infrared (FT-IR) spectroscopy coupled with multivariate statistical analyses was used for sub-typing 30 strains of E. coli O157:H7 that had previously been typed by multilocus variable number tandem repeat analysis (MLVA) and pulsed field gel electrophoresis (PFGE). Hierarchical cluster analysis (HCA) and canonical variate analysis (CVA) of the FT-IR spectra resulted in the clustering of the same or similar MLVA types and separation of different MLVA types of E. coli O157:H7. The developed FT-IR method showed better discriminatory power than PFGE in sub-typing E. coli O157:H7. Results also indicated the spectral relatedness between different outbreak strains. However, the grouping of some strains was not in complete agreement with the clustering based on PFGE and MLVA. Additionally, HCA of the spectra differentiated the strains into 30 sub-clusters, indicating the high specificity and suitability of the method for strain level identification. Strains were also classified (97% correct) based on the type of Shiga toxin present using CVA of the spectra. This study demonstrated that FT-IR spectroscopy is suitable for rapid (≤16 h) and economical sub-typing of E. coli O157:H7 with comparable accuracy to MLVA typing. This is the first report of using an FT-IR-based method for sub-typing E. coli O157:H7. Copyright © 2012 Elsevier Ltd. All rights reserved.

Increased glycosylation efficiency of recombinant proteins in Escherichia coli by auto-induction.

PubMed

Ding, Ning; Yang, Chunguang; Sun, Shenxia; Han, Lichi; Ruan, Yao; Guo, Longhua; Hu, Xuejun; Zhang, Jianing

2017-03-25

Escherichia coli cells have been considered as promising hosts for producing N-glycosylated proteins since the successful production of N-glycosylated protein in E. coli with the pgl (N-linked protein glycosylation) locus from Campylobacter jejuni. However, one hurdle in producing N-glycosylated proteins in large scale using E. coli is inefficient glycan glycosylation. In this study, we developed a strategy for the production of N-glycosylated proteins with high efficiency via an optimized auto-induction method. The 10th human fibronectin type III domain (FN3) was engineered with native glycosylation sequon DFNRSK and optimized DQNAT sequon in C-terminus with flexible linker as acceptor protein models. The resulting glycosylation efficiencies were confirmed by Western blots with anti-FLAG M1 antibody. Increased efficiency of glycosylation was obtained by changing the conventional IPTG induction to auto-induction method, which increased the glycosylation efficiencies from 60% and 75% up to 90% and 100% respectively. Moreover, in the condition of inserting the glycosylation sequon in the loop of FN3 (the acceptor sequon with local structural conformation), the glycosylation efficiency was increased from 35% to 80% by our optimized auto-induction procedures. To justify the potential for general application of the optimized auto-induction method, the reconstituted lsg locus from Haemophilus influenzae and PglB from C. jejuni were utilized, and this led to 100% glycosylation efficiency. Our studies provided quantitative evidence that the optimized auto-induction method will facilitate the large-scale production of pure exogenous N-glycosylation proteins in E. coli cells. Copyright © 2017 Elsevier Inc. All rights reserved.

A comparative study of low pH stress in E. coli and S. typhimurium, and a comparative study of the inducibility of lysyl-tRNA synthetase in the enterobacteriaceae

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

Hickey, E.W.

1988-01-01

Lysyl-tRNA synthetase (LRS) in Escherichia coli is coded by two genes, one constitutive, and the other inducible. The commonness of inducibility of this enzyme in prokaryotes was first tested in eight members of the Enterobacteriaceae using culture conditions known to induce it in E. coli. LRS was found to be inducible in Salmonella Typhimurium, Citrobacter freundii, Klebsiella pneumoniae and Enterobacter aerogenes, but not in Serratia marcescens, Proteus mirabilis, Proteus vulgaris or Morganella morganii. The results also indicated that LRS was not induced in E. coli grown in defined medium (SMM) at an external pH (pH{sub 0}) of 5.0, whereas, itmore » was induced in S. typhimurium under this condition. Further investigation of low pH{sub 0} induced behavior in E. coli and S. typhimurium by quantitation of H{sub 2} {sup 35}SO{sub 4} labeled proteins from two dimensional polyacrylamide gels of whole cell sonic extracts showed that at least twenty proteins were induced from 2- to 16-fold in S. typhimurium grown at pH{sub 0} 5.0 or shifted from growth at pH{sub 0} 7.0 to 5.0. Internal pH (pH{sub i}) changes occurring during steady state growth at low pH{sub 0}, and on shifting from pH{sub 0} 7.0 to 5.0, were measured using {sup 14}C-benzoic acid uptake.« less

Rapid amperometric detection of Escherichia coli in wastewater by measuring β-D glucuronidase activity with disposable carbon sensors.

PubMed

Rochelet, Murielle; Solanas, Sébastien; Betelli, Laetitia; Chantemesse, Benoît; Vienney, Fabienne; Hartmann, Alain

2015-09-10

An assay on the indirect amperometric quantification of the β-D-Glucuronidase (GLUase) activity was developed for the rapid and specific detection of Escherichia coli (E. coli) in complex environmental samples. The p-aminophenyl β-D-glucopyranoside (PAPG) was selected as an electrochemical substrate for GLUase measurement and the p-aminophenol (PAP) released during the enzymatic hydrolysis was monitored by cyclic voltammetry with disposable carbon screen-printed sensors. The intensity of the measured anodic peak current was proportional to the amount of GLUase, and therefore to the number of E. coli in the tested sample. Once the substrate concentration and pH values optimized, a GLUase detection limit of 10 ng mL(-1) was achieved. Using a procedure involving a filtration step of the bacteria followed by their incubation with the substrate solution containing both the nonionic detergent Triton X-100 as permeabilization agent and the culture media Luria broth to monitor the growth, filtered bacterial cells ranging from 5 × 10(4) to 10(8) UFC/membrane were detected within 3 h. The amperometric assay was applied to the determination of fecal contamination in raw and treated wastewater samples and it was successfully compared with conventional bacterial plating methods and uidA gene quantitative PCR. Owing to its ability to perform measurements in turbid media, the GLUase amperometric method is a reliable tool for the rapid and decentralized quantification of viable but also nonculturable E. coli in complex environmental samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Constrained Allocation Flux Balance Analysis

PubMed Central

Mori, Matteo; Hwa, Terence; Martin, Olivier C.

2016-01-01

New experimental results on bacterial growth inspire a novel top-down approach to study cell metabolism, combining mass balance and proteomic constraints to extend and complement Flux Balance Analysis. We introduce here Constrained Allocation Flux Balance Analysis, CAFBA, in which the biosynthetic costs associated to growth are accounted for in an effective way through a single additional genome-wide constraint. Its roots lie in the experimentally observed pattern of proteome allocation for metabolic functions, allowing to bridge regulation and metabolism in a transparent way under the principle of growth-rate maximization. We provide a simple method to solve CAFBA efficiently and propose an “ensemble averaging” procedure to account for unknown protein costs. Applying this approach to modeling E. coli metabolism, we find that, as the growth rate increases, CAFBA solutions cross over from respiratory, growth-yield maximizing states (preferred at slow growth) to fermentative states with carbon overflow (preferred at fast growth). In addition, CAFBA allows for quantitatively accurate predictions on the rate of acetate excretion and growth yield based on only 3 parameters determined by empirical growth laws. PMID:27355325

Highly diverse variable number tandem repeat loci in the E. coli O157:H7 and O55:H7 genomes for high-resolution molecular typing.

PubMed

Keys, C; Kemper, S; Keim, P

2005-01-01

Evaluation of the Escherichia coli genome for variable number tandem repeat (VNTR) loci in order to provide a subtyping tool with greater discrimination and more efficient capacity. Twenty-nine putative VNTR loci were identified from the E. coli genomic sequence. Their variability was validated by characterizing the number of repeats at each locus in a set of 56 E. coli O157:H7/HN and O55:H7 isolates. An optimized multiplex assay system was developed to facility high capacity analysis. Locus diversity values ranged from 0.23 to 0.95 while the number of alleles ranged from two to 29. This multiple-locus VNTR analysis (MLVA) data was used to describe genetic relationships among these isolates and was compared with PFGE (pulse field gel electrophoresis) data from a subset of the same strains. Genetic similarity values were highly correlated between the two approaches, through MLVA was capable of discrimination amongst closely related isolates when PFGE similar values were equal to 1.0. Highly variable VNTR loci exist in the E. coli O157:H7 genome and are excellent estimators of genetic relationships, in particular for closely related isolates. Escherichia coli O157:H7 MLVA offers a complimentary analysis to the more traditional PFGE approach. Application of MLVA to an outbreak cluster could generate superior molecular epidemiology and result in a more effective public health response.

Fabrication, Characterization, and Evaluation of Bionanocomposites Based on Natural Polymers and Antibiotics for Wound Healing Applications.

PubMed

Rădulescu, Marius; Holban, Alina Maria; Mogoantă, Laurențiu; Bălşeanu, Tudor-Adrian; Mogoșanu, George Dan; Savu, Diana; Popescu, Roxana Cristina; Fufă, Oana; Grumezescu, Alexandru Mihai; Bezirtzoglou, Eugenia; Lazar, Veronica; Chifiriuc, Mariana Carmen

2016-06-10

The aim of our research activity was to obtain a biocompatible nanostructured composite based on naturally derived biopolymers (chitin and sodium alginate) loaded with commercial antibiotics (either Cefuroxime or Cefepime) with dual functions, namely promoting wound healing and assuring the local delivery of the loaded antibiotic. Compositional, structural, and morphological evaluations were performed by using the thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and fourier transform infrared spectroscopy (FTIR) analytical techniques. In order to quantitatively and qualitatively evaluate the biocompatibility of the obtained composites, we performed the tetrazolium-salt (MTT) and agar diffusion in vitro assays on the L929 cell line. The evaluation of antimicrobial potential was evaluated by the viable cell count assay on strains belonging to two clinically relevant bacterial species (i.e., Escherichia coli and Staphylococcus aureus).

Comparative analysis of Escherichia coli O157 growth and protein-expression, in vitro and in vivo, in rumen fluid of cattle

USDA-ARS?s Scientific Manuscript database

Cattle are the primary reservoirs for Escherichia coli O157 (O157), a Shiga toxin-producing E. coli, with potential for serious extraintestinal sequelae in humans. In a recent study (Kudva IT et al. BMC Microbiol. 2014; 14:48), we reported that when cultured in rumen fluid from dairy cattle on the ...

Genomic and transcriptomic analysis of Escherichia coli strains associated with persistent and transient bovine mastitis and the role of colanic acid

USDA-ARS?s Scientific Manuscript database

Escherichia coli is a leading cause of bacterial mastitis in dairy cattle. This infection is most often transient in nature, causing an infection that lasts 2–3 days. However, E. coli has been shown to cause a persistent infection in a minority of cases. The mechanisms that allow for a persistent E....

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.

Minimum inhibitory (MIC) and minimum microbicidal concentration (MMC) of polihexanide and triclosan against antibiotic sensitive and resistant Staphylococcus aureus and Escherichia coli strains

PubMed Central

Assadian, Ojan; Wehse, Katrin; Hübner, Nils-Olaf; Koburger, Torsten; Bagel, Simone; Jethon, Frank; Kramer, Axel

2011-01-01

Background: An in-vitro study was conducted investigating the antimicrobial efficacy of polihexanide and triclosan against clinical isolates and reference laboratory strains of Staphylococcus aureus and Escherichia coli. Methods: The minimal inhibitory concentration (MIC) and the minimal microbicidal concentration (MMC) were determined following DIN 58940-81 using a micro-dilution assay and a quantitative suspension test following EN 1040. Polihexanide was tested in polyethylene glycol 4000, triclosan in aqueous solutions. Results: Against all tested strains the MIC of polihexanide ranged between 1–2 µg/mL. For triclosan the MICs varied depending on strains ranging between 0.5 µg/mL for the reference strains and 64 µg/mL for two clinical isolates. A logRF >5 without and logRF >3 with 0.2% albumin burden was achieved at 0.6 µg/mL triclosan. One exception was S. aureus strain H-5-24, where a triclosan concentration of 0.6 µg/mL required 1 minute without and 10 minutes with albumin burden to achieve the same logRFs. Polihexanide achieved a logRF >5 without and logRF >3 with albumin burden at a concentration of 0.6 µg/mL within 30 sec. The exception was the North-German epidemic MRSA strain, were an application time of 5 minutes was required. Conclusion: The clinical isolates of E. coli generally showed higher MICs against triclosan, both in the micro-dilution assay as well in the quantitative suspension test than comparable reference laboratory strains. For polihexanide and triclosan strain dependant susceptibility was shown. However, both antimicrobial compounds are effective when used in concentrations common in practice. PMID:22242087

General Platform for Systematic Quantitative Evaluation of Small-Molecule Permeability in Bacteria

PubMed Central

2015-01-01

The chemical features that impact small-molecule permeability across bacterial membranes are poorly understood, and the resulting lack of tools to predict permeability presents a major obstacle to the discovery and development of novel antibiotics. Antibacterials are known to have vastly different structural and physicochemical properties compared to nonantiinfective drugs, as illustrated herein by principal component analysis (PCA). To understand how these properties influence bacterial permeability, we have developed a systematic approach to evaluate the penetration of diverse compounds into bacteria with distinct cellular envelopes. Intracellular compound accumulation is quantitated using LC-MS/MS, then PCA and Pearson pairwise correlations are used to identify structural and physicochemical parameters that correlate with accumulation. An initial study using 10 sulfonyladenosines in Escherichia coli, Bacillus subtilis, and Mycobacterium smegmatis has identified nonobvious correlations between chemical structure and permeability that differ among the various bacteria. Effects of cotreatment with efflux pump inhibitors were also investigated. This sets the stage for use of this platform in larger prospective analyses of diverse chemotypes to identify global relationships between chemical structure and bacterial permeability that would enable the development of predictive tools to accelerate antibiotic drug discovery. PMID:25198656

A probabilistic QMRA of Salmonella in direct agricultural reuse of treated municipal wastewater.

PubMed

Amha, Yamrot M; Kumaraswamy, Rajkumari; Ahmad, Farrukh

2015-01-01

Developing reliable quantitative microbial risk assessment (QMRA) procedures aids in setting recommendations on reuse applications of treated wastewater. In this study, a probabilistic QMRA to determine the risk of Salmonella infections resulting from the consumption of edible crops irrigated with treated wastewater was conducted. Quantitative polymerase chain reaction (qPCR) was used to enumerate Salmonella spp. in post-disinfected samples, where they showed concentrations ranging from 90 to 1,600 cells/100 mL. The results were used to construct probabilistic exposure models for the raw consumption of three vegetables (lettuce, cabbage, and cucumber) irrigated with treated wastewater, and to estimate the disease burden using Monte Carlo analysis. The results showed elevated median disease burden, when compared with acceptable disease burden set by the World Health Organization, which is 10⁻⁶ disability-adjusted life years per person per year. Of the three vegetables considered, lettuce showed the highest risk of infection in all scenarios considered, while cucumber showed the lowest risk. The results of the Salmonella concentration obtained with qPCR were compared with the results of Escherichia coli concentration for samples taken on the same sampling dates.

Quantitative assessment of the association between APC promoter methylation and breast cancer.

PubMed

He, Keli; Zhang, Li; Long, Xinghua

2016-06-21

Adenomatous polyposis coli (APC) is an important tumor suppressor gene in breast cancer. However, there were inconsistent conclusions in the association between APC promoter methylation and breast cancer. Hence, we conducted a meta-analysis to quantitatively assess the clinicopathological significance and diagnosis role of APC methylation in breast cancer. In total, 3172 samples from 29 studies were performed in this study. The odds ratio (OR) of APC methylation was 5.92 (95% CI = 3.16-11.07) in breast cancer cases compared to controls,. The APC promoter methylation was associated with cancer stage (OR = 0.47, 95% CI = 0.28-0.80, P = 0.006), lymph node metastases (OR = 0.55, 95% CI = 0.36-0.84, P = 0.005) and ER status (OR = 1.34, 95% CI = 1.03-1.73, P = 0.003) in breast cancer. Furthermore, the sensitivity and specificity for all included studies were 0.444 (95% CI: 0.321-0.575, P < 0.0001) and 0.976 (95% CI: 0.916-0.993, P < 0.0001), respectively. These results suggested that APC promoter methylation was associated with breast cancer risk, and it could be a valuable biomarker for diagnosis, treatment and prognosis of breast cancer.

Reconstructing genome-wide regulatory network of E. coli using transcriptome data and predicted transcription factor activities

PubMed Central

2011-01-01

Background Gene regulatory networks play essential roles in living organisms to control growth, keep internal metabolism running and respond to external environmental changes. Understanding the connections and the activity levels of regulators is important for the research of gene regulatory networks. While relevance score based algorithms that reconstruct gene regulatory networks from transcriptome data can infer genome-wide gene regulatory networks, they are unfortunately prone to false positive results. Transcription factor activities (TFAs) quantitatively reflect the ability of the transcription factor to regulate target genes. However, classic relevance score based gene regulatory network reconstruction algorithms use models do not include the TFA layer, thus missing a key regulatory element. Results This work integrates TFA prediction algorithms with relevance score based network reconstruction algorithms to reconstruct gene regulatory networks with improved accuracy over classic relevance score based algorithms. This method is called Gene expression and Transcription factor activity based Relevance Network (GTRNetwork). Different combinations of TFA prediction algorithms and relevance score functions have been applied to find the most efficient combination. When the integrated GTRNetwork method was applied to E. coli data, the reconstructed genome-wide gene regulatory network predicted 381 new regulatory links. This reconstructed gene regulatory network including the predicted new regulatory links show promising biological significances. Many of the new links are verified by known TF binding site information, and many other links can be verified from the literature and databases such as EcoCyc. The reconstructed gene regulatory network is applied to a recent transcriptome analysis of E. coli during isobutanol stress. In addition to the 16 significantly changed TFAs detected in the original paper, another 7 significantly changed TFAs have been detected by using our reconstructed network. Conclusions The GTRNetwork algorithm introduces the hidden layer TFA into classic relevance score-based gene regulatory network reconstruction processes. Integrating the TFA biological information with regulatory network reconstruction algorithms significantly improves both detection of new links and reduces that rate of false positives. The application of GTRNetwork on E. coli gene transcriptome data gives a set of potential regulatory links with promising biological significance for isobutanol stress and other conditions. PMID:21668997

Alignment-free design of highly discriminatory diagnostic primer sets for Escherichia coli O104:H4 outbreak strains.

PubMed

Pritchard, Leighton; Holden, Nicola J; Bielaszewska, Martina; Karch, Helge; Toth, Ian K

2012-01-01

An Escherichia coli O104:H4 outbreak in Germany in summer 2011 caused 53 deaths, over 4000 individual infections across Europe, and considerable economic, social and political impact. This outbreak was the first in a position to exploit rapid, benchtop high-throughput sequencing (HTS) technologies and crowdsourced data analysis early in its investigation, establishing a new paradigm for rapid response to disease threats. We describe a novel strategy for design of diagnostic PCR primers that exploited this rapid draft bacterial genome sequencing to distinguish between E. coli O104:H4 outbreak isolates and other pathogenic E. coli isolates, including the historical hæmolytic uræmic syndrome (HUSEC) E. coli HUSEC041 O104:H4 strain, which possesses the same serotype as the outbreak isolates. Primers were designed using a novel alignment-free strategy against eleven draft whole genome assemblies of E. coli O104:H4 German outbreak isolates from the E. coli O104:H4 Genome Analysis Crowd-Sourcing Consortium website, and a negative sequence set containing 69 E. coli chromosome and plasmid sequences from public databases. Validation in vitro against 21 'positive' E. coli O104:H4 outbreak and 32 'negative' non-outbreak EHEC isolates indicated that individual primer sets exhibited 100% sensitivity for outbreak isolates, with false positive rates of between 9% and 22%. A minimal combination of two primers discriminated between outbreak and non-outbreak E. coli isolates with 100% sensitivity and 100% specificity. Draft genomes of isolates of disease outbreak bacteria enable high throughput primer design and enhanced diagnostic performance in comparison to traditional molecular assays. Future outbreak investigations will be able to harness HTS rapidly to generate draft genome sequences and diagnostic primer sets, greatly facilitating epidemiology and clinical diagnostics. We expect that high throughput primer design strategies will enable faster, more precise responses to future disease outbreaks of bacterial origin, and help to mitigate their societal impact.

A globotetraosylceramide (Gb₄) receptor-based ELISA for quantitative detection of Shiga toxin 2e.

PubMed

Togashi, Katsuhiro; Sasaki, Shiho; Sato, Wataru

2015-08-01

Currently, no simple assays are available for routine quantitative detection of Escherichia coli-produced Shiga toxin 2e (Stx2e) that causes porcine edema disease. Here, we present a novel quantitative detection method for Stx2e based on the measurement of Stx2e binding to the specific globotetraosylceramide (Gb4) receptor by ELISA (Gb4-ELISA). No cross-reactivity was found with the other Shiga toxins Stx1 and Stx2, indicating high specificity. When the recombinant Stx2e B subunit (Stx2eB) was used, the absorbance measured by Gb4-ELISA increased linearly with Stx2eB concentration in the range of 20-2,500 ng/ml. The Gb4-ELISA method can be easily performed, suggesting that it would be a useful diagnostic tool for porcine edema disease.

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.

Mechanisms of iron regulation of luminescence in Vibrio fischeri.

PubMed Central

Haygood, M G; Nealson, K H

1985-01-01

Synthesis of luciferase (an autoinducible enzyme) is repressed by iron in the symbiotic bioluminescent bacterium Vibrio fischeri. Possible mechanisms of iron regulation of luciferase synthesis were tested with V. fischeri and with Escherichia coli clones containing plasmids carrying V. fischeri luminescence genes. Experiments were conducted in complete medium with and without the synthetic iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid). Comparison of the effect of ethylenediamine-di(o-hydroxyphenyl acetic acid) and another growth inhibitor, (2-n-heptyl-4-hydroxyquinoline-N-oxide), showed that iron repression is not due to inhibition of growth. A quantitative bioassay for autoinducer was developed with E. coli HB101 containing pJE411, a plasmid carrying V. fischeri luminescence genes with a transcriptional fusion between luxI and E. coli lacZ. Bioassay experiments showed no effect of iron on either autoinducer activity or production (before induction) or transcription of the lux operon. Ethylenediamine-di(o-hydroxyphenyl acetic acid) did not affect luciferase induction in E. coli strains with wild-type iron assimilation (ED8654) or impaired iron assimilation (RW193) bearing pJE202 (a plasmid with functional V. fischeri lux genes), suggesting that the genes responsible for the iron effect are missing or substituted in these clones. Two models are consistent with the data: (i) iron represses autoinducer transport, and (ii) iron acts through an autoinduction-independent regulatory system (e.g., an iron repressor). PMID:3920202

Action of Escherichia coli Enterotoxin: Adenylate Cyclase Behavior of Intestinal Epithelial Cells in Culture

PubMed Central

Kantor, Harvey S.; Tao, Pearl; Wisdom, Charlene

1974-01-01

Heat-labile enterotoxin preparations obtained from two enteropathogenic strains of Escherichia coli of porcine and human origin were shown to stimulate adenylate cyclase activity of human embryonic intestinal epithelial cells in culture. Comparable results were also obtained when cholera toxin was used. The degree of enzyme stimulation was proportional to the concentration of enterotoxin. Similar preparations from two strains of non-enterotoxigenic E. coli had no effect on adenylate cyclase activity. Cells exposed to enterotoxin could be washed after 1 min of contact time without altering the subsequent course of maximum adenylate cyclase activity, which was maintained for at least 18 h at 37 C. During long periods (18 h) of tissue culture incubation, the determination of adenylate cyclase activity was 200- to 300-fold more sensitive than quantitating fluid accumulation in the adult rabbit ileal loop model. Decreasing the incubation time appreciably reduced the sensitivity of the epithelial cells to enterotoxin. E. coli enterotoxin is an effective activator of nonintestinal adenylate cyclase systems. Treatment of KB and HEp-2 cell lines with enterotoxin also resulted in significant enzyme stimulation. The intestinal epithelial cell tissue culture model provides a sensitive homogenous biological system for studying the response of intestinal adenylate cyclase to enterotoxin while eliminating the numerous cellular and tissue components present in the ligated ileal loop model. PMID:4364505

Rapid and high resolution genotyping of all Escherichia coli serotypes using 10 genomic repeat-containing loci.

PubMed

Løbersli, Inger; Haugum, Kjersti; Lindstedt, Bjørn-Arne

2012-01-01

Our laboratory has previously published two multiple-locus variable-number tandem-repeats analysis (MLVA) methods for rapid genotyping of Escherichia coli (E. coli), which are now in routine use for surveillance and outbreak detection. The first assay developed was specific for E. coli O157:H7; however this assay was not suitable for genotyping other E. coli serotypes. A new generic MLVA-assay was then developed with the capability of genotyping all E. coli serotypes. This generic E. coli MLVA (GECM7) was based on polymorphism in seven variable number of tandem repeats (VNTR) loci. GECM7 worked well with the majority of E. coli serotypes; however we wanted to increase the resolution for this method based in part of comparison with PFGE typing of E. coli O26:H11, where PFGE appeared to display higher resolution. The GECM7 method was improved by adding three new repeat-loci to a total of ten (GECM10), and a considerable increase in resolution was observed (from 296 to 507 genotypes on the same set of strains). Copyright © 2011 Elsevier B.V. All rights reserved.

Black soldier fly (Diptera: Stratiomyidae) larvae reduce Escherichia coli in dairy manure.

PubMed

Liu, Qiaolin; Tomberlin, Jeffery K; Brady, Jeff A; Sanford, Michelle R; Yu, Ziniu

2008-12-01

Escherichia coli labeled with a green fluorescent protein was inoculated into sterile dairy manure at 7.0 log cfu/g. Approximately 125 black soldier fly larvae were placed in manure inoculated and homogenized with E. coli. Manure inoculated with E. coli but without black soldier fly larvae served as the control. For the first experiment, larvae were introduced into 50, 75, 100, or 125 g sterilized dairy manure inoculated and homogenized with E. coli and stored 72 h at 27 degrees C. Black soldier fly larvae significantly reduced E. coli counts in all treatments. However, varying the amount of manure provided the black soldier fly larvae significantly affected their weight gain and their ability to reduce E. coli populations present. For the second experiment, larvae were introduced into 50 g manure inoculated with E. coli and stored for 72 h at 23, 27, 31, or 35 degrees C. Minimal bacterial growth was recorded in the control held at 35 degrees C and was excluded from the analysis. Black soldier fly larvae significantly reduced E. coli counts in manure held at remaining temperatures. Accordingly, temperature significantly influenced the ability of black soldier fly larvae to develop and reduce E. coli counts with greatest suppression occurring at 27 degrees C.

Identification, cloning and sequencing of Escherichia coli strain chi1378 (O78:K80) iss gene isolated from poultry colibacillosis in Iran.

PubMed

Derakhshandeh, A; Zahraei Salehi, T; Tadjbakhsh, H; Karimi, V

2009-09-01

To identify, clone and sequence the iss (increased serum survival) gene from E. coli strain chi1378 isolated from Iranian poultry and to predict its protein product, Iss. The iss gene from E. coli strain chi1378 was amplified and cloned into the pTZ57R/T vector and sequenced. From the DNA sequence, the Iss predictive protein was evaluated using bioinformatics. Iss from strain chi1378 had 100% identity with other E. coli serotypes and isolates from different origins and also 98% identity with E. coli O157:H7 Iss protein. Phylogenetic analysis showed no significant different phylogenic groups among E. coli strains. The strong association of predicted Iss protein among different E. coli strains suggests that it could be a good antigen to control and detect avian pathogenic E. coli (APEC). Because the exact pathogenesis and the role of virulence factors are unknown, the Iss protein could be used as a target for vaccination in the future, but further research is required.

Slugs: potential novel vectors of Escherichia coli O157.

PubMed

Sproston, Emma L; Macrae, M; Ogden, Iain D; Wilson, Michael J; Strachan, Norval J C

2006-01-01

Field and laboratory studies were performed to determine whether slugs could act as novel vectors for pathogen (e.g., Escherichia coli O157) transfer from animal feces to salad vegetables. Escherichia coli O157 was isolated from 0.21% of field slugs from an Aberdeenshire sheep farm. These isolates carried the verocytotoxin genes (vt1 and vt2) and the attaching and effacing gene (eae), suggesting that they are potentially pathogenic to humans. Strain typing using multilocus variable number tandem repeats analysis showed that slug and sheep isolates were indistinguishable. Laboratory experiments using an E. coli mutant resistant to nalidixic acid showed that the ubiquitous slug species Deroceras reticulatum could carry viable E. coli on its external surface for up to 14 days. Slugs that had been fed E. coli shed viable bacteria in their feces with numbers showing a short but statistically significant linear log decline. Further, it was found that E. coli persisted for up to 3 weeks in excreted slug feces, and hence, we conclude that slugs have the potential to act as novel vectors of E. coli O157.

Slugs: Potential Novel Vectors of Escherichia coli O157

PubMed Central

Sproston, Emma L.; Macrae, M.; Ogden, Iain D.; Wilson, Michael J.; Strachan, Norval J. C.

2006-01-01

Field and laboratory studies were performed to determine whether slugs could act as novel vectors for pathogen (e.g., Escherichia coli O157) transfer from animal feces to salad vegetables. Escherichia coli O157 was isolated from 0.21% of field slugs from an Aberdeenshire sheep farm. These isolates carried the verocytotoxin genes (vt1 and vt2) and the attaching and effacing gene (eae), suggesting that they are potentially pathogenic to humans. Strain typing using multilocus variable number tandem repeats analysis showed that slug and sheep isolates were indistinguishable. Laboratory experiments using an E. coli mutant resistant to nalidixic acid showed that the ubiquitous slug species Deroceras reticulatum could carry viable E. coli on its external surface for up to 14 days. Slugs that had been fed E. coli shed viable bacteria in their feces with numbers showing a short but statistically significant linear log decline. Further, it was found that E. coli persisted for up to 3 weeks in excreted slug feces, and hence, we conclude that slugs have the potential to act as novel vectors of E. coli O157. PMID:16391036

Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH).

PubMed

Almeida, Carina; Azevedo, Nuno F; Santos, Sílvio; Keevil, Charles W; Vieira, Maria J

2011-03-29

Our current understanding of biofilms indicates that these structures are typically composed of many different microbial species. However, the lack of reliable techniques for the discrimination of each population has meant that studies focusing on multi-species biofilms are scarce and typically generate qualitative rather than quantitative data. We employ peptide nucleic acid fluorescence in situ hybridization (PNA FISH) methods to quantify and visualize mixed biofilm populations. As a case study, we present the characterization of Salmonella enterica/Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven different support materials. Ex-situ, we were able to monitor quantitatively the populations of ∼56 mixed species biofilms up to 48 h, regardless of the support material. In situ, a correct quantification remained more elusive, but a qualitative understanding of biofilm structure and composition is clearly possible by confocal laser scanning microscopy (CLSM) at least up to 192 h. Combining the data obtained from PNA FISH/CLSM with data from other established techniques and from calculated microbial parameters, we were able to develop a model for this tri-species biofilm. The higher growth rate and exopolymer production ability of E. coli probably led this microorganism to outcompete the other two [average cell numbers (cells/cm(2)) for 48 h biofilm: E. coli 2,1 × 10(8) (± 2,4 × 10(7)); L. monocytogenes 6,8 × 10(7) (± 9,4 × 10(6)); and S. enterica 1,4 × 10(6) (± 4,1 × 10(5))]. This overgrowth was confirmed by CSLM, with two well-defined layers being easily identified: the top one with E. coli, and the bottom one with mixed regions of L. monocytogenes and S. enterica. While PNA FISH has been described previously for the qualitative study of biofilm populations, the present investigation demonstrates that it can also be used for the accurate quantification and spatial distribution of species in polymicrobial communities. Thus, it facilitates the understanding of interspecies interactions and how these are affected by changes in the surrounding environment.

Changes in Escherichia coli to Cryptosporidium ratios for various fecal pollution sources and drinking water intakes.

PubMed

Lalancette, Cindy; Papineau, Isabelle; Payment, Pierre; Dorner, Sarah; Servais, Pierre; Barbeau, Benoit; Di Giovanni, George D; Prévost, Michèle

2014-05-15

Assessing the presence of human pathogenic Cryptosporidium oocysts in surface water remains a significant water treatment and public health challenge. Most drinking water suppliers rely on fecal indicators, such as the well-established Escherichia coli (E. coli), to avoid costly Cryptosporidium assays. However, the use of E. coli has significant limitations in predicting the concentration, the removal and the transport of Cryptosporidium. This study presents a meta-analysis of E. coli to Cryptosporidium concentration paired ratios to compare their complex relationships in eight municipal wastewater sources, five agricultural fecal pollution sources and at 13 drinking water intakes (DWI) to a risk threshold based on US Environmental Protection Agency (USEPA) regulations. Ratios lower than the USEPA risk threshold suggested higher concentrations of oocysts in relation to E. coli concentrations, revealing an underestimed risk for Cryptosporidium based on E. coli measurements. In raw sewage (RS), high ratios proved E. coli (or fecal coliforms) concentrations were a conservative indicator of Cryptosporidium concentrations, which was also typically true for secondary treated wastewater (TWW). Removals of fecal indicator bacteria (FIB) and parasites were quantified in WWTPs and their differences are put forward as a plausible explanation of the sporadic ratio shift. Ratios measured from agricultural runoff surface water were typically lower than the USEPA risk threshold and within the range of risk misinterpretation. Indeed, heavy precipitation events in the agricultural watershed led to high oocyst concentrations but not to E. coli or enterococci concentrations. More importantly, ratios established in variously impacted DWI from 13 Canadian drinking water plants were found to be related to dominant fecal pollution sources, namely municipal sewage. In most cases, when DWIs were mainly influenced by municipal sewage, E. coli or fecal coliforms concentrations agreed with Cryptosporidium concentrations as estimated by the meta-analysis, but when DWIs were influenced by agricultural runoff or wildlife, there was a poor relationship. Average recovery values were available for 6 out of 22 Cryptosporidium concentration data sets and concomitant analysis demonstrated no changes in trends, with and without correction. Nevertheless, recovery assays performed along with every oocyst count would have enhanced the precision of this work. Based on our findings, the use of annual averages of E. coli concentrations as a surrogate for Cryptosporidium concentrations can result in an inaccurate estimate of the Cryptosporidium risk for agriculture impacted drinking water intakes or for intakes with more distant wastewater sources. Studies of upstream fecal pollution sources are recommended for drinking water suppliers to improve their interpretation of source water quality data. Copyright © 2014 Elsevier Ltd. All rights reserved.

Adenovirus-mediated in utero gene transfer in mice and guinea pigs: tissue distribution of recombinant adenovirus determined by quantitative TaqMan-polymerase chain reaction assay.

PubMed

Senoo, M; Matsubara, Y; Fujii, K; Nagasaki, Y; Hiratsuka, M; Kure, S; Uehara, S; Okamura, K; Yajima, A; Narisawa, K

2000-04-01

Fetal somatic cell gene therapy could become an attractive solution for some congenital genetic diseases or the disorders which manifest themselves during the fetal period. We performed adenovirus-mediated gene transfer to mice and guinea pig fetuses in utero and evaluated the efficiency of gene transfer by histochemical analysis and a quantitative TaqMan-polymerase chain reaction (TaqMan-PCR) assay. We first injected a replication-deficient recombinant adenovirus containing the Escherichia coli LacZ gene driven by a CAG promoter (AxCALacZ) into pregnant mice through the amniotic space, placenta, or intraperitoneal space of the fetus. Histochemical analysis showed limited transgene expression in fetal tissues. We then administered AxCALacZ to guinea pig fetuses in the late stage of pregnancy through the umbilical vein. The highest beta-galactosidase expression was observed in liver followed by moderate expression in heart, spleen, and adrenal gland. The transgene expression was also present in kidney, intestine, and placenta to a lesser degree. No positively stained cells were observed in lung, muscle, or pancreas except in the vascular endothelium of these organs. Quantitative measurement of recombinant adenoviral DNA by the TaqMan-PCR assay showed that the vast majority of the injected viruses was present in liver. The current study indicated that adenovirus-mediated gene transfer into guinea pig fetus through the umbilical vein is feasible and results in efficient transgene expression in fetal tissues. The experimental procedures using pregnant guinea pigs might serve as a good experimental model for in utero gene transfer. Since our TaqMan-PCR assay detects the LacZ gene, one of the most widely used reporter genes, it may be generally applicable to adenovirus quantification in various gene transfer experiments.

NMR-based metabolomic urinalysis: a rapid screening test for urinary tract infection.

PubMed

Lam, Ching-Wan; Law, Chun-Yiu; To, Kelvin Kai-Wang; Cheung, Stanley Kwok-Kuen; Lee, Kim-Chung; Sze, Kong-Hung; Leung, Ka-Fai; Yuen, Kwok-Yung

2014-09-25

Urinary tract infection (UTI) is one of the most common bacterial infections in humans; however, there is no accurate and fast quantitative test to detect UTI. Dipstick urinalysis is semi-quantitative with a limited diagnostic accuracy, while urine culture is accurate but takes time. We described a quantitative biochemical method for the diagnosis of bacteriuria using a single marker. We compared the urine metabolomes from 88 patients with bacterial UTI and 61 controls using (1)H NMR spectroscopy followed by principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). The biomarker identified was subsequently validated using independent samples. The urine acetic acid/creatinine (mmol/mmol) level was determined to be the most discriminatory marker for bacterial UTI with an area-under-receiver operating characteristic curve=0.97, sensitivity=91% and specificity=95% at the optimal cutoff 0.03 mmol/mmol. For validation, 60 samples were recruited prospectively. Using the optimal cutoff for acetic acid/creatinine, this method showed sensitivity=96%, specificity=94%, positive predictive value=92%, negative predictive value=97% and an overall accuracy=95%. The diagnostic performance was superior to dipstick urinalysis or microscopy. In addition, we also observed an increase of urinary trimethylamine (TMA) in patients with Escherichia coli-associated UTI. TMA is a mammalian-microbial co-metabolite and the high level of TMA generated is related to the bacterial enzyme, trimethylamine N-oxide (TMAO) reductase which reduces TMAO to TMA. Urine acetic acid is a neglected metabolite that can be used for rapid diagnosis of UTI and TMA can be used for etiologic diagnosis of UTI. With the introduction of NMR-based clinical analyzers to clinical laboratories, NMR-based urinalysis can be translated for clinical use. Copyright © 2014 Elsevier B.V. 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.

Mathematical model of rhamnolipid production using E.coli bacteria

NASA Astrophysics Data System (ADS)

Adham, Muhammad Fariduddin; Apri, Mochamad; Moeis, Maelita Ramdani

2018-03-01

Rhamnolipid is one of biosurfactants that is widely used in many industries. Despite its wide use, production of rhamnolipid usually involves a pathogen that may endanger our health. To tackle this issue, in iGEM (International Genetically Engineered Machine) competition 2015, our team engineered Escherichia coli (E.coli) to produce rhamnolipid. The bacteria were then put into medium containing glucose and lactose. It turned out that bacteria E. coli produced lower rhamnolipid than that by pseudomonas, therefore a good strategy is required to improve their productivity. We present a mathematical model to describe the production of rhamnolipid by the engineered E coli. Using bifurcation analysis, the equilibrium points of the model and their stabilities were analyzed as the amount of lactose was varied. We show that the system produces bistability behavior for some interval values of lactose. From this analysis we found that to guarantee a high production of rhamnolipid, a high level of lactose is required. To maintain the productivity, however, it is sufficient to maintain the lactose level above a certain threshold value.

Biofilm formation and sanitizer resistance of Escherichia coli 0157:H7 strains isolated from "High Event Period" meat contamination

USDA-ARS?s Scientific Manuscript database

In the meat industry, a “High Event Period” (HEP) is defined as a time period during which commercial meat plants experience a higher than usual rate of E. coli O157:H7 contamination. Genetic analysis indicated that within a HEP, most of the E. coli O157:H7 strains belong to a singular dominant str...

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

Voluntarily exposure to a single, high dose of probiotic Escherichia coli results in prolonged colonisation.

PubMed

Wassenaar, T M; Beimfohr, C; Geske, T; Zimmermann, K

2014-12-01

The ability of probiotic Escherichia coli to colonise the human gut was determined in a volunteer study following national (German) regulations. Five persons voluntarily took a single, high dose of Symbioflor®2, which contains 6 different probiotic E. coli genotypes, to assess tolerance of the product, after which presence of E. coli in their faeces was tested for a follow-up period of 30 weeks. Intake of the product did not result in severe side effect in any of the individuals, though mild side effects were observed. Stool analysis showed that the probiotic E. coli had colonised all five persons for a period of 10 to 30 weeks (mean: 18.7 weeks, median: 25.7 weeks). In two individuals there was evidence of competition between host E. coli and probiotic E. coli, while in two others total E. coli levels increased persistently with at least a factor of 10 as a result of the received dose. In one individual, who had lacked detectable levels of faecal E. coli at the start of the post-authorisation safety study, long-term colonisation was established, first by probiotic E. coli exclusively, which were later replaced by host E. coli strains. In four out of five individuals, total E. coli faecal counts were higher on average than at the start of the experiment, while in none total levels exceeded 5×107 cfu/g. When the specific genotypes of the 6 probiotic E. coli were analysed, it was found that one and the same common genotype was responsible for prolonged colonisation in all five individuals.

Synergy between (13)C-metabolic flux analysis and flux balance analysis for understanding metabolic adaptation to anaerobiosis in E. coli.

PubMed

Chen, Xuewen; Alonso, Ana P; Allen, Doug K; Reed, Jennifer L; Shachar-Hill, Yair

2011-01-01

Genome-based Flux Balance Analysis (FBA) and steady-state isotopic-labeling-based Metabolic Flux Analysis (MFA) are complimentary approaches to predicting and measuring the operation and regulation of metabolic networks. Here, genome-derived models of Escherichia coli (E. coli) metabolism were used for FBA and ¹³C-MFA analyses of aerobic and anaerobic growths of wild-type E. coli (K-12 MG1655) cells. Validated MFA flux maps reveal that the fraction of maintenance ATP consumption in total ATP production is about 14% higher under anaerobic (51.1%) than aerobic conditions (37.2%). FBA revealed that an increased ATP utilization is consumed by ATP synthase to secrete protons from fermentation. The TCA cycle is shown to be incomplete in aerobically growing cells and submaximal growth is due to limited oxidative phosphorylation. An FBA was successful in predicting product secretion rates in aerobic culture if both glucose and oxygen uptake measurement were constrained, but the most-frequently predicted values of internal fluxes yielded from sampling the feasible space differ substantially from MFA-derived fluxes. © 2010 Elsevier Inc. All rights reserved.

Profiling of composition and metabolic activities of the colonic microflora of growing pigs fed diets supplemented with prebiotic oligosaccharides.

PubMed

Mountzouris, Konstantinos C; Balaskas, Christos; Fava, Fransesca; Tuohy, Kieran M; Gibson, Glenn R; Fegeros, K

2006-08-01

It is evident that quantitative information on different microbial groups and their contribution in terms of activity in the gastrointestinal (GI) tract of humans and animals is required in order to formulate functional diets targeting improved gut function and host health. In this work, quantitative information on levels and spatial distributions of Bacteroides spp, Eubacterium spp, Clostridium spp, Escherichia coli, Bifidobacterium spp and Lactobacillus/Enterococcus spp. along the porcine large intestine was investigated using 16S rRNA targeted probes and fluorescent in situ hybridisation (FISH). Caecum, ascending colon (AC) and rectum luminal digesta from three groups of individually housed growing pigs fed either a corn-soybean basal diet (CON diet) or a prebiotic diet containing 10 g/kg oligofructose (FOS diet) or trans-galactooligosaccharides (TOS diet) at the expense of cornstarch were analysed. DAPI staining was used to enumerate total number of cells in the samples. Populations of total cells, Bacteroides, Eubacterium, Clostridium and Bifidobacterium declined significantly (P < 0.05) from caecum to rectum, and were not affected by dietary treatments. Populations of Lactobacillus/Enterococcus and E. coli did not differ throughout the large intestine. The relative percent (%) contribution of each bacterial group to the total cell count did not differ between caecum and rectum, with the exception of Eubacterium that was higher in the AC digesta. FISH analysis showed that the sum of all bacterial groups made up a small percentage of the total cells, which was 12.4%, 21.8% and 10.3% in caecum, AC and rectum, respectively. This supports the view that in swine, the diversity of GI microflora might be higher compared to other species. In terms of microflora metabolic activity, the substantially higher numerical trends seen in FOS and TOS treatments regarding total volatile fatty acid, acetate concentrations and glycolytic activities, it could be postulated that FOS and TOS promoted saccharolytic activities in the porcine colon.

Histomorphometric characteristics of immune cells in small intestine of pigs perorally immunized with vaccine candidate F18ac nonenterotoxigenic E. coli strain.

PubMed

Kovšca Janjatović, A; Lacković, G; Božić, F; Spoljarić, D; Popović, M; Valpotić, H; Vijtiuk, N; Pavičić, Z; Valpotić, I

2009-12-29

Colidiarrhea and colienterotoxemia caused by F4(+) and/or F18(+) enterotoxigenic E. coli (ETEC) strains are the most prevalent infections of suckling and weaned pigs. Here we tested the immunogenicity and protective effectiveness of attenuated F18ac(+) non-ETEC vaccine candidate strain against challenge infection with F4ac(+) ETEC strain by quantitative phenotypic analysis of small intestinal leukocyte subsets in weaned pigs.We also evaluated levamisole as an immune response modifier (IRM) and its adjuvanticity when given in the combination with the experimental vaccine. The pigs were parenterally immunized with either levamisole (at days -2, -1 and 0) or with levamisole and perorally given F18ac(+) non-ETEC strain (at day 0), and challenged with F4ac(+) ETEC strain 7 days later.At day 13 the pigs were euthanatized and sampled for immunohistological/histomorphometrical analyses. Lymphoid CD3(+), CD45RA(+), CD45RC(+), CD21(+), IgA(+) and myeloid SWC3(+) cell subsets were identified in jejunal and ileal epithelium, lamina propria and Peyer's patches using the avidin-biotin complex method, and their numbers were determined by computer-assisted histomorphometry. Quantitative immunophenotypic analyses showed that levamisole treated pigs had highly increased numbers of jejunal CD3(+), CD45RC(+) and SWC3(+) cells (p<0.05) as compared to those recorded in nontreated control pigs.In the ileum of these pigs we have recorded that only CD21(+) cells were significantly increased (p<0.01). The pigs that were treated with levamisole adjuvanted experimental vaccine had significantly increased numbers of all tested cell subsets in both segments of the small intestine. It was concluded that levamisole adjuvanted F18ac(+) non-ETEC vaccine was a requirement for the elicitation of protective gut immunity in this model; nonspecific immunization with levamisole was less effective, but confirmed its potential as an IRM.

Programmable living material containing reporter micro-organisms permits quantitative detection of oligosaccharides.

PubMed

Mora, Carlos A; Herzog, Antoine F; Raso, Renzo A; Stark, Wendelin J

2015-08-01

The increasing molecular understanding of many diseases today permits the development of new diagnostic methods. However, few easy-to-handle and inexpensive tools exist for common diseases such as food disorders. Here we present a living material based analytical sensor (LiMBAS) containing genetically modified bacteria (Escherichia coli) immobilized and protected in a thin layer between a nanoporous and support polymer membrane for a facile quantification of disease-relevant oligosaccharides. The bacteria were engineered to fluoresce in response to the analyte to reveal its diffusion behavior when using a blue-light source and optical filter. We demonstrated that the diffusion zone diameter was related semi-logarithmically to the analyte concentration. LiMBAS could accurately quantify lactose or galactose in undiluted food samples and was able to measure food intolerance relevant concentrations in the range of 1-1000 mM requiring a sample volume of 1-10 μL. LiMBAS was storable for at least seven days without losing functionality at 4 °C. A wide range of genetic tools for E. coli are readily available thus allowing the reprogramming of the material to serve as biosensor for other molecules. In combination with smartphones, an automated diagnostic analysis becomes feasible which would also allow untrained people to use LiMBAS. Copyright © 2015 Elsevier Ltd. All rights reserved.

Virulence profiling and quantification of verocytotoxin-producing Escherichia coli O145:H28 and O26:H11 isolated during an ice cream-related hemolytic uremic syndrome outbreak.

PubMed

Buvens, Glenn; Possé, Björn; De Schrijver, Koen; De Zutter, Lieven; Lauwers, Sabine; Piérard, Denis

2011-03-01

In September-October 2007, a mixed-serotype outbreak of verocytotoxin-producing Escherichia coli (VTEC) O145:H28 and O26:H11 occurred in the province of Antwerp, Belgium. Five girls aged between 2 and 11 years developed hemolytic uremic syndrome, and seven other coexposed persons with bloody diarrhea were identified. Laboratory confirmation of O145:H28 infection was obtained for three hemolytic uremic syndrome patients, one of whom was coinfected with O26:H11. The epidemiological and laboratory investigations revealed ice cream as the most likely source of the outbreak. The ice cream was produced at a local dairy farm using pasteurized milk. VTEC of both serotypes with indistinguishable pulsed-field gel electrophoresis patterns were isolated from patients, ice cream, and environmental samples. Quantitative analysis of the ice cream indicated concentrations of 2.4 and 0.03 CFU/g for VTEC O145 and O26, respectively. Virulence typing revealed that the repertoire of virulence genes carried by the O145:H28 outbreak strain was comparable to that of O157 VTEC and more exhaustive as compared to the O26:H11 outbreak strain and nonrelated clinical strains belonging to these serotypes. Taken together, these data suggest that O145:H28 played the most important role in this outbreak.

[Cloning and sequence analysis of recombinant fusion gene of Escherichia coli heat-liable enterotoxin B subunit and Actinobacillus actinomycetemcomitans fimbria associative protein].

PubMed

Li, Yi; Sun, Hong-chen; Guo, Xue-jun; Feng, Shu-zhang

2005-02-01

To clone the recombinant fusion gene of Escherichia coli heat-liable enterotoxin B subunit (Ltb) and Actinobacillus actinomycetemcomitans fimbria associative protein (Fap). Two couples of primers were designed for PCR according to the known sequence of ltb and fap. The ltb and fap gene were obtained by amplification PCR technique from plasmid EWD299 of Escherichia coli and Actinobacillus actinomycetemcomitans 310 DNA respectively, and fused them by PCR. The fusion gene ltb-fap were cloning into plasmid pET28a(+). The recombined plasmid pET28a ltb-fap was transformed into Escherichia coli DH5alpha. The recombinant was screened and identified by restriction enzyme and PCR. The cloned gene was sequenced. The ltb-fap about 531bp in size was obtained successfully, and identified by PCR, restrictive enzyme and sequence analysis. The vector of pET28a ltb-fap was obtained.

Risk of Transmission of Antimicrobial Resistant Escherichia coli from Commercial Broiler and Free-Range Retail Chicken in India

PubMed Central

Hussain, Arif; Shaik, Sabiha; Ranjan, Amit; Nandanwar, Nishant; Tiwari, Sumeet K.; Majid, Mohammad; Baddam, Ramani; Qureshi, Insaf A.; Semmler, Torsten; Wieler, Lothar H.; Islam, Mohammad A.; Chakravortty, Dipshikha; Ahmed, Niyaz

2017-01-01

Multidrug-resistant Escherichia coli infections are a growing public health concern. This study analyzed the possibility of contamination of commercial poultry meat (broiler and free-range) with pathogenic and or multi-resistant E. coli in retail chain poultry meat markets in India. We analyzed 168 E. coli isolates from broiler and free-range retail poultry (meat/ceca) sampled over a wide geographical area, for their antimicrobial sensitivity, phylogenetic groupings, virulence determinants, extended-spectrum-β-lactamase (ESBL) genotypes, fingerprinting by Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR and genetic relatedness to human pathogenic E. coli using whole genome sequencing (WGS). The prevalence rates of ESBL producing E. coli among broiler chicken were: meat 46%; ceca 40%. Whereas, those for free range chicken were: meat 15%; ceca 30%. E. coli from broiler and free-range chicken exhibited varied prevalence rates for multi-drug resistance (meat 68%; ceca 64% and meat 8%; ceca 26%, respectively) and extraintestinal pathogenic E. coli (ExPEC) contamination (5 and 0%, respectively). WGS analysis confirmed two globally emergent human pathogenic lineages of E. coli, namely the ST131 (H30-Rx subclone) and ST117 among our poultry E. coli isolates. These results suggest that commercial poultry meat is not only an indirect public health risk by being a possible carrier of non-pathogenic multi-drug resistant (MDR)-E. coli, but could as well be the carrier of human E. coli pathotypes. Further, the free-range chicken appears to carry low risk of contamination with antimicrobial resistant and extraintestinal pathogenic E. coli (ExPEC). Overall, these observations reinforce the understanding that poultry meat in the retail chain could possibly be contaminated by MDR and/or pathogenic E. coli. PMID:29180984

Risk of Transmission of Antimicrobial Resistant Escherichia coli from Commercial Broiler and Free-Range Retail Chicken in India.

PubMed

Hussain, Arif; Shaik, Sabiha; Ranjan, Amit; Nandanwar, Nishant; Tiwari, Sumeet K; Majid, Mohammad; Baddam, Ramani; Qureshi, Insaf A; Semmler, Torsten; Wieler, Lothar H; Islam, Mohammad A; Chakravortty, Dipshikha; Ahmed, Niyaz

2017-01-01

Multidrug-resistant Escherichia coli infections are a growing public health concern. This study analyzed the possibility of contamination of commercial poultry meat (broiler and free-range) with pathogenic and or multi-resistant E. coli in retail chain poultry meat markets in India. We analyzed 168 E. coli isolates from broiler and free-range retail poultry (meat/ceca) sampled over a wide geographical area, for their antimicrobial sensitivity, phylogenetic groupings, virulence determinants, extended-spectrum-β-lactamase (ESBL) genotypes, fingerprinting by Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR and genetic relatedness to human pathogenic E. coli using whole genome sequencing (WGS). The prevalence rates of ESBL producing E. coli among broiler chicken were: meat 46%; ceca 40%. Whereas, those for free range chicken were: meat 15%; ceca 30%. E. coli from broiler and free-range chicken exhibited varied prevalence rates for multi-drug resistance (meat 68%; ceca 64% and meat 8%; ceca 26%, respectively) and extraintestinal pathogenic E. coli (ExPEC) contamination (5 and 0%, respectively). WGS analysis confirmed two globally emergent human pathogenic lineages of E. coli , namely the ST131 ( H 30-Rx subclone) and ST117 among our poultry E. coli isolates. These results suggest that commercial poultry meat is not only an indirect public health risk by being a possible carrier of non-pathogenic multi-drug resistant (MDR)- E. coli , but could as well be the carrier of human E. coli pathotypes. Further, the free-range chicken appears to carry low risk of contamination with antimicrobial resistant and extraintestinal pathogenic E. coli (ExPEC). Overall, these observations reinforce the understanding that poultry meat in the retail chain could possibly be contaminated by MDR and/or pathogenic E. coli.

Estimating the size of the solution space of metabolic networks

PubMed Central

Braunstein, Alfredo; Mulet, Roberto; Pagnani, Andrea

2008-01-01

Background Cellular metabolism is one of the most investigated system of biological interactions. While the topological nature of individual reactions and pathways in the network is quite well understood there is still a lack of comprehension regarding the global functional behavior of the system. In the last few years flux-balance analysis (FBA) has been the most successful and widely used technique for studying metabolism at system level. This method strongly relies on the hypothesis that the organism maximizes an objective function. However only under very specific biological conditions (e.g. maximization of biomass for E. coli in reach nutrient medium) the cell seems to obey such optimization law. A more refined analysis not assuming extremization remains an elusive task for large metabolic systems due to algorithmic limitations. Results In this work we propose a novel algorithmic strategy that provides an efficient characterization of the whole set of stable fluxes compatible with the metabolic constraints. Using a technique derived from the fields of statistical physics and information theory we designed a message-passing algorithm to estimate the size of the affine space containing all possible steady-state flux distributions of metabolic networks. The algorithm, based on the well known Bethe approximation, can be used to approximately compute the volume of a non full-dimensional convex polytope in high dimensions. We first compare the accuracy of the predictions with an exact algorithm on small random metabolic networks. We also verify that the predictions of the algorithm match closely those of Monte Carlo based methods in the case of the Red Blood Cell metabolic network. Then we test the effect of gene knock-outs on the size of the solution space in the case of E. coli central metabolism. Finally we analyze the statistical properties of the average fluxes of the reactions in the E. coli metabolic network. Conclusion We propose a novel efficient distributed algorithmic strategy to estimate the size and shape of the affine space of a non full-dimensional convex polytope in high dimensions. The method is shown to obtain, quantitatively and qualitatively compatible results with the ones of standard algorithms (where this comparison is possible) being still efficient on the analysis of large biological systems, where exact deterministic methods experience an explosion in algorithmic time. The algorithm we propose can be considered as an alternative to Monte Carlo sampling methods. PMID:18489757

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.

Secretome analysis of diarrhea-inducing strains of Escherichia coli

PubMed Central

Nirujogi, Raja Sekhar; Muthusamy, Babylakshmi; Kim, Min-Sik; Sathe, Gajanan J.; Lakshmi, P.T.V.; Kovbasnjuk, Olga N.; Prasad, T.S. Keshava; Wade, Mary; Jabbour, Rabih E.

2017-01-01

Secreted proteins constitute a major part of virulence factors that are responsible for pathogenesis caused by Gram-negative bacteria. Enterohemorrhagic Escherichia coli, O157:H7, is the major pathogen often causing outbreaks. However, studies have reported that the significant outbreaks caused by non-O157:H7 E. coli strains, also known as “Big-Six” serogroup strains, are increasing. There is no systematic study describing differential secreted proteins from these non-O157:H7 E. coli strains. In this study, we carried out MS-based differential secretome analysis using tandem mass tags labeling strategy of non-O157:H7 E. coli strains, O103, O111, O121, O145, O26, and O45. We identified 1241 proteins, of which 565 proteins were predicted to be secreted. We also found that 68 proteins were enriched in type III secretion system and several of them were differentially expressed across the strains. Additionally, we identified several strain-specific secreted proteins that could be used for developing potential markers for the identification and strain-level differentiation. To our knowledge, this study is the first comparative proteomic study on secretome of E. coli Big-Six serogroup and the several of these strain-specific secreted proteins can be further studied to develop potential markers for identification and strain-level differentiation. Moreover, the results of this study can be utilized in several applications, including food safety, diagnostics of E. coli outbreaks, and detection and identification of bio threats in biodefense. PMID:28070933

Expression and purification of the antimicrobial peptide GSL1 in bacteria for raising antibodies.

PubMed

Meiyalaghan, Sathiyamoorthy; Latimer, Julie M; Kralicek, Andrew V; Shaw, Martin L; Lewis, John G; Conner, Anthony J; Barrell, Philippa J

2014-11-04

The Gibberellin Stimulated-Like (GSL) or Snakin peptides from higher plants are cysteine-rich, with broad spectrum activity against a range of bacterial and fungal pathogens. To detect GSL peptides in applications such as western blot analysis and enzyme-linked immunosorbent assays (ELISA), specific antibodies that recognise GSL peptides are required. However, the intrinsic antimicrobial activity of these peptides is likely to prevent their expression alone in bacterial or yeast expression systems for subsequent antibody production in animal hosts. To overcome this issue we developed an Escherichia coli expression strategy based on the expression of the GSL1 peptide as a His-tagged thioredoxin fusion protein. The DNA sequence for the mature GSL1 peptide from potato (Solanum tuberosum L.) was cloned into the pET-32a expression vector to produce a construct encoding N-terminally tagged his6-thioredoxin-GSL1. The fusion protein was overexpressed in E. coli to produce soluble non-toxic protein. The GSL1 fusion protein could be easily purified by using affinity chromatography to yield ~1.3 mg of his6-thioredoxin-GSL1 per L of culture. The fusion protein was then injected into rabbits for antibody production. Western blot analysis showed that the antibodies obtained from rabbit sera specifically recognised the GSL1 peptide that had been expressed in a wheat germ cell-free expression system. We present here the first report of a GSL1 peptide expressed as a fusion protein with thioredoxin that has resulted in milligram quantities of soluble protein to be produced. We have also demonstrated that a wheat germ system can be used to successfully express small quantities of GSL1 peptide useful as positive control in western blot analysis. To our knowledge this is the first report of antibodies being produced against GSL1 peptide. The antibodies will be useful for analysis of GSL1peptides in western blot, localization by immunohistochemistry (IHC) and quantitation by ELISA.

Rapid screening of the antimicrobial efficacy of Ag zeolites.

PubMed

Tosheva, L; Belkhair, S; Gackowski, M; Malic, S; Al-Shanti, N; Verran, J

2017-09-01

A semi-quantitative screening method was used to compare the killing efficacy of Ag zeolites against bacteria and yeast as a function of the zeolite type, crystal size and concentration. The method, which substantially reduced labor, consumables and waste and provided an excellent preliminary screen, was further validated by quantitative plate count experiments. Two pairs of zeolite X and zeolite beta with different sizes (ca. 200nm and 2μm for zeolite X and ca. 250 and 500nm for zeolite beta) were tested against Escherichia coli (E. coli) and Candida albicans (C. albicans) at concentrations in the range 0.05-0.5mgml -1 . Reduction of the zeolite crystal size resulted in a decrease in the killing efficacy against both microorganisms. The semi-quantitative tests allowed convenient optimization of the zeolite concentrations to achieve targeted killing times. Zeolite beta samples showed higher activity compared to zeolite X despite their lower Ag content, which was attributed to the higher concentration of silver released from zeolite beta samples. Cytotoxicity measurements using peripheral blood mononuclear cells (PBMCs) indicated that Ag zeolite X was more toxic than Ag zeolite beta. However, the trends for the dependence of cytotoxicity on zeolite crystal size at different zeolite concentrations were different for the two zeolites and no general conclusions about zeolite cytotoxicity could be drawn from these experiments. This result indicates a complex relationship, requiring the necessity for individual cytotoxicity measurements for all antimicrobial applications based on the use of zeolites. Copyright © 2017 Elsevier B.V. All rights reserved.

Chronic Sequelae of E. coli O157: Systematic Review and Meta-analysis of the Proportion of E. coli O157 Cases That Develop Chronic Sequelae

PubMed Central

Sargeant, Jan; Thomas, M. Kate; Fazil, Aamir

2014-01-01

Abstract Objective: This was a systematic review and meta-analysis to determine the proportion of Escherichia coli O157 cases that develop chronic sequelae. Data Sources: We conducted a systematic review of articles published prior to July 2011 in Pubmed, Agricola, CabDirect, or Food Safety and Technology Abstracts. Study Selection: Studies were selected that reported the number of E. coli O157 cases that developed reactive arthritis (ReA), hemolytic uremic syndrome (HUS), irritable bowel syndrome, inflammatory bowel disease, or Guillain Barré syndrome. Methods: Three levels of screening and data extraction of articles were conducted using predefined data fields. Meta-analysis was performed on unique outcome measures using a random-effects model, and heterogeneity was assessed using the I2 value. Meta-regression was used to explore the influence of nine study-level variables on heterogeneity. Results: A total of 82 studies were identified reporting 141 different outcome measures; 81 reported on HUS and one reported on ReA. Depending on the number of cases of E. coli O157, the estimate for the proportion of E. coli O157 cases that develop HUS ranged from 17.2% in extra-small studies (<50 cases) to 4.2% in extra-large studies (>1000 cases). Heterogeneity was significantly associated with group size (p<0.0001); however, the majority of the heterogeneity was unexplained. Conclusions: High unexplained heterogeneity indicated that the study-level factors examined had a minimal influence on the variation of estimates reported. PMID:24404780

Campylobacter Immunity and Quantitative Excretion Rates in Thai Children

DTIC Science & Technology

1993-01-01

vitro studies suggest that other pathogens, in- excluded if the diarrhea had lasted >3 days or jfthe patient had C 0 ~~~~~~~recently received...antibiotics. Age.smtsadciclancluding Shigella and Salmonella species and Escherichia coli. . . symptoms. and clinical and treatment data were recorded, and...ubiq- static cellulitic lesions progressing to bullae formation, skin uitous in the estuarine environment. It can cause a syndrome necrosis, necrotizin

Thermotolerant coliforms are not a good surrogate for Campylobacter spp. in environmental water.

PubMed

St-Pierre, Karen; Lévesque, Simon; Frost, Eric; Carrier, Nathalie; Arbeit, Robert D; Michaud, Sophie

2009-11-01

This study aimed to assess the importance of quantitatively detecting Campylobacter spp. in environmental surface water. The prevalence and the quantity of Campylobacter spp., thermotolerant coliforms, and Escherichia coli in 2,471 samples collected weekly, over a 2-year period, from 13 rivers and 12 streams in the Eastern Townships, Québec, Canada, were determined. Overall, 1,071 (43%), 1,481 (60%), and 1,463 (59%) samples were positive for Campylobacter spp., thermotolerant coliforms, and E. coli, respectively. There were weak correlations between the weekly distributions of Campylobacter spp. and thermotolerant coliforms (Spearman's rho coefficient = 0.27; P = 0.008) and between the quantitative levels of the two classes of organisms (Kendall tau-b correlation coefficient = 0.233; P < 0.0001). Well water samples from the Eastern Townships were also tested. Five (10%) of 53 samples from private surface wells were positive for Campylobacter jejuni, of which only 2 were positive for thermotolerant coliforms. These findings suggest that microbial monitoring of raw water by using only fecal indicator organisms is not sufficient for assessing the occurrence or the load of thermophilic Campylobacter spp. Insights into the role of environmental water as sources for sporadic Campylobacter infection will require genus-specific monitoring techniques.

Precision Sensing by Two Opposing Gradient Sensors: How Does Escherichia coli Find its Preferred pH Level?

PubMed Central

Hu, Bo; Tu, Yuhai

2013-01-01

It is essential for bacteria to find optimal conditions for their growth and survival. The optimal levels of certain environmental factors (such as pH and temperature) often correspond to some intermediate points of the respective gradients. This requires the ability of bacteria to navigate from both directions toward the optimum location and is distinct from the conventional unidirectional chemotactic strategy. Remarkably, Escherichia coli cells can perform such a precision sensing task in pH taxis by using the same chemotaxis machinery, but with opposite pH responses from two different chemoreceptors (Tar and Tsr). To understand bacterial pH sensing, we developed an Ising-type model for a mixed cluster of opposing receptors based on the push-pull mechanism. Our model can quantitatively explain experimental observations in pH taxis for various mutants and wild-type cells. We show how the preferred pH level depends on the relative abundance of the competing sensors and how the sensory activity regulates the behavioral response. Our model allows us to make quantitative predictions on signal integration of pH and chemoattractant stimuli. Our study reveals two general conditions and a robust push-pull scheme for precision sensing, which should be applicable in other adaptive sensory systems with opposing gradient sensors. PMID:23823247

Micro-patterned agarose gel devices for single-cell high-throughput microscopy of E. coli cells.

PubMed

Priest, David G; Tanaka, Nobuyuki; Tanaka, Yo; Taniguchi, Yuichi

2017-12-21

High-throughput microscopy of bacterial cells elucidated fundamental cellular processes including cellular heterogeneity and cell division homeostasis. Polydimethylsiloxane (PDMS)-based microfluidic devices provide advantages including precise positioning of cells and throughput, however device fabrication is time-consuming and requires specialised skills. Agarose pads are a popular alternative, however cells often clump together, which hinders single cell quantitation. Here, we imprint agarose pads with micro-patterned 'capsules', to trap individual cells and 'lines', to direct cellular growth outwards in a straight line. We implement this micro-patterning into multi-pad devices called CapsuleHotel and LineHotel for high-throughput imaging. CapsuleHotel provides ~65,000 capsule structures per mm 2 that isolate individual Escherichia coli cells. In contrast, LineHotel provides ~300 line structures per mm that direct growth of micro-colonies. With CapsuleHotel, a quantitative single cell dataset of ~10,000 cells across 24 samples can be acquired and analysed in under 1 hour. LineHotel allows tracking growth of > 10 micro-colonies across 24 samples simultaneously for up to 4 generations. These easy-to-use devices can be provided in kit format, and will accelerate discoveries in diverse fields ranging from microbiology to systems and synthetic biology.

Modeling of scale-dependent bacterial growth by chemical kinetics approach.

PubMed

Martínez, Haydee; Sánchez, Joaquín; Cruz, José-Manuel; Ayala, Guadalupe; Rivera, Marco; Buhse, Thomas

2014-01-01

We applied the so-called chemical kinetics approach to complex bacterial growth patterns that were dependent on the liquid-surface-area-to-volume ratio (SA/V) of the bacterial cultures. The kinetic modeling was based on current experimental knowledge in terms of autocatalytic bacterial growth, its inhibition by the metabolite CO2, and the relief of inhibition through the physical escape of the inhibitor. The model quantitatively reproduces kinetic data of SA/V-dependent bacterial growth and can discriminate between differences in the growth dynamics of enteropathogenic E. coli, E. coli JM83, and Salmonella typhimurium on one hand and Vibrio cholerae on the other hand. Furthermore, the data fitting procedures allowed predictions about the velocities of the involved key processes and the potential behavior in an open-flow bacterial chemostat, revealing an oscillatory approach to the stationary states.

Physical Sensing of Surface Properties by Microswimmers--Directing Bacterial Motion via Wall Slip.

PubMed

Hu, Jinglei; Wysocki, Adam; Winkler, Roland G; Gompper, Gerhard

2015-05-20

Bacteria such as Escherichia coli swim along circular trajectories adjacent to surfaces. Thereby, the orientation (clockwise, counterclockwise) and the curvature depend on the surface properties. We employ mesoscale hydrodynamic simulations of a mechano-elastic model of E. coli, with a spherocylindrical body propelled by a bundle of rotating helical flagella, to study quantitatively the curvature of the appearing circular trajectories. We demonstrate that the cell is sensitive to nanoscale changes in the surface slip length. The results are employed to propose a novel approach to directing bacterial motion on striped surfaces with different slip lengths, which implies a transformation of the circular motion into a snaking motion along the stripe boundaries. The feasibility of this approach is demonstrated by a simulation of active Brownian rods, which also reveals a dependence of directional motion on the stripe width.

Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli

PubMed Central

Beal, Jacob; Haddock-Angelli, Traci; Gershater, Markus; de Mora, Kim; Lizarazo, Meagan; Hollenhorst, Jim; Rettberg, Randy

2016-01-01

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices. PMID:26937966

3M™ Molecular detection system versus MALDI-TOF mass spectrometry and molecular techniques for the identification of Escherichia coli 0157:H7, Salmonella spp. &Listeria spp.

PubMed

Loff, Marché; Mare, Louise; de Kwaadsteniet, Michele; Khan, Wesaal

2014-06-01

The aim of this study was to compare standard selective plating, conventional PCR (16S rRNA and species specific primers), MALDI-TOF MS and the 3M™ Molecular Detection System for the routine detection of the pathogens Listeria, Salmonella and Escherichia coli 0157:H7 in wastewater and river water samples. MALDI-TOF MS was able to positively identify 20/21 (95%) of the E. coli isolates obtained at genus and species level, while 16S rRNA sequencing only correctly identified 6/21 (28%) as E. coli strains. None of the presumptive positive Listeria spp. and Salmonella spp. isolates obtained by culturing on selective media were positively identified by MALDI-TOF and 16S rRNA analysis. The species-specific E. coli 0157:H7 PCR described in this present study, was not able to detect any E. coli 0157:H7 strains in the wastewater and river water samples analysed. However, E. coli strains, Listeria spp., L. monocytogenes and Salmonella spp. were detected using species specific PCR. Escherichia coli 0157:H7, Listeria spp. and Salmonella spp. were also sporadically detected throughout the sampling period in the wastewater and river water samples analysed by the 3M™ Molecular Detection System. MALDI-TOF MS, which is a simple, accurate and cost-effective detection method, efficiently identified the culturable organisms, while in the current study both species specific PCR (Listeria spp. and Salmonella spp.) and 3M™ Molecular Detection System could be utilised for the direct routine analysis of pathogens in water sources. Copyright © 2014 Elsevier B.V. All rights reserved.

Post mortem Survival of Gallibacterium anatis in a Laying Hen Experimental Infection Model.

PubMed

Wang, Chong; Pors, Susanne Elisabeth; Bojesen, Anders Miki

2018-06-01

To assess the survival of Gallibacterium anatis in dead laying hens, 21-wk-old laying hens were injected intraperitoneally with 0.5 ml brain hearth infusion broth containing 10 8 colony-forming units (CFU) of G. anatis 12656-12 liver ( n = 16), Escherichia coli ST141 ( n = 16), or a mix of G. anatis 12656-12 liver and E. coli ST141 ( n = 16), respectively. Birds were euthanatized 24 hr post injection. From each group eight dead birds were kept at 4 C and eight at room temperature. Swab samples were taken at different time points post euthanatization and streaked on blood agar plates. From the birds kept at 4 C, G. anatis was reisolated from the G. anatis and the G. anatis- E. coli co-injected groups at least 12 days post euthanization. From birds kept at room temperature, G. anatis was reisolated up to 2 days post euthanatization. When using the gyrB-based G. anatis-specific quantitative PCR (qPCR), G. anatis was detected within at least 5 days, and up to 5 days post euthanatization, from birds kept at room temperature and 4 C, respectively. Escherichia coli was reisolated from all the time points independent of how the birds were kept. No difference was observed between the reisolation rates for G. anatis or E. coli when comparing similar detection methods. For birds kept at 4 C, bacterial cultivation was a more sensitive method for detecting G. anatis ( P < 0.05), whereas for birds kept at room temperature, the G. anatis-specific qPCR outperformed bacterial culture ( P < 0.05). In conclusion, we demonstrated that G. anatis has a poorer survival rate than does E. coli in dead chickens kept at room temperature. That finding may affect the overall diagnostic sensitivity and lead to underdiagnosis of G. anatis in a normal production setting.

Polymorphic Variation in Susceptibility and Metabolism of Triclosan-Resistant Mutants of Escherichia coli and Klebsiella pneumoniae Clinical Strains Obtained after Exposure to Biocides and Antibiotics

PubMed Central

Curiao, Tânia; Marchi, Emmanuela; Viti, Carlo; Oggioni, Marco R.; Baquero, Fernando; Martinez, José Luis

2015-01-01

Exposure to biocides may result in cross-resistance to other antimicrobials. Changes in biocide and antibiotic susceptibilities, metabolism, and fitness costs were studied here in biocide-selected Escherichia coli and Klebsiella pneumoniae mutants. E. coli and K. pneumoniae mutants with various degrees of triclosan susceptibility were obtained after exposure to triclosan (TRI), benzalkonium chloride (BKC), chlorhexidine (CHX) or sodium hypochlorite (SHC), and ampicillin or ciprofloxacin. Alterations in antimicrobial susceptibility and metabolism in mutants were tested using Phenotype MicroArrays. The expression of AcrAB pump and global regulators (SoxR, MarA, and RamA) was measured by quantitative reverse transcription-PCR (qRT-PCR), and the central part of the fabI gene was sequenced. The fitness costs of resistance were assessed by a comparison of relative growth rates. Triclosan-resistant (TRIr) and triclosan-hypersusceptible (TRIhs) mutants of E. coli and K. pneumoniae were obtained after selection with biocides and/or antibiotics. E. coli TRIr mutants, including those with mutations in the fabI gene or in the expression of acrB, acrF, and marA, exhibited changes in susceptibility to TRI, CHX, and antibiotics. TRIr mutants for which the TRI MIC was high presented improved metabolism of carboxylic acids, amino acids, and carbohydrates. In TRIr mutants, resistance to one antimicrobial provoked hypersusceptibility to another one(s). TRIr mutants had fitness costs, particularly marA-overexpressing (E. coli) or ramA-overexpressing (K. pneumoniae) mutants. TRI, BKC, and CIP exposure frequently yielded TRIr mutants exhibiting alterations in AraC-like global regulators (MarA, SoxR, and RamA), AcrAB-TolC, and/or FabI, and influencing antimicrobial susceptibility, fitness, and metabolism. These various phenotypes suggest a trade-off of different selective processes shaping the evolution toward antibiotic/biocide resistance and influencing other adaptive traits. PMID:25824225

Polymorphic variation in susceptibility and metabolism of triclosan-resistant mutants of Escherichia coli and Klebsiella pneumoniae clinical strains obtained after exposure to biocides and antibiotics.

PubMed

Curiao, Tânia; Marchi, Emmanuela; Viti, Carlo; Oggioni, Marco R; Baquero, Fernando; Martinez, José Luis; Coque, Teresa M

2015-01-01

Exposure to biocides may result in cross-resistance to other antimicrobials. Changes in biocide and antibiotic susceptibilities, metabolism, and fitness costs were studied here in biocide-selected Escherichia coli and Klebsiella pneumoniae mutants. E. coli and K. pneumoniae mutants with various degrees of triclosan susceptibility were obtained after exposure to triclosan (TRI), benzalkonium chloride (BKC), chlorhexidine (CHX) or sodium hypochlorite (SHC), and ampicillin or ciprofloxacin. Alterations in antimicrobial susceptibility and metabolism in mutants were tested using Phenotype MicroArrays. The expression of AcrAB pump and global regulators (SoxR, MarA, and RamA) was measured by quantitative reverse transcription-PCR (qRT-PCR), and the central part of the fabI gene was sequenced. The fitness costs of resistance were assessed by a comparison of relative growth rates. Triclosan-resistant (TRI(r)) and triclosan-hypersusceptible (TRI(hs)) mutants of E. coli and K. pneumoniae were obtained after selection with biocides and/or antibiotics. E. coli TRI(r) mutants, including those with mutations in the fabI gene or in the expression of acrB, acrF, and marA, exhibited changes in susceptibility to TRI, CHX, and antibiotics. TRI(r) mutants for which the TRI MIC was high presented improved metabolism of carboxylic acids, amino acids, and carbohydrates. In TRI(r) mutants, resistance to one antimicrobial provoked hypersusceptibility to another one(s). TRI(r) mutants had fitness costs, particularly marA-overexpressing (E. coli) or ramA-overexpressing (K. pneumoniae) mutants. TRI, BKC, and CIP exposure frequently yielded TRI(r) mutants exhibiting alterations in AraC-like global regulators (MarA, SoxR, and RamA), AcrAB-TolC, and/or FabI, and influencing antimicrobial susceptibility, fitness, and metabolism. These various phenotypes suggest a trade-off of different selective processes shaping the evolution toward antibiotic/biocide resistance and influencing other adaptive traits. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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.

Simultaneous detection of Escherichia coli O157:H7 and Salmonella Typhimurium using quantum dots as fluorescence labels.

PubMed

Yang, Liju; Li, Yanbin

2006-03-01

In this study, we explored the use of semiconductor quantum dots (QDs) as fluorescence labels in immunoassays for simultaneous detection of two species of foodborne pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium. QDs with different sizes can be excited with a single wavelength of light, resulting in different emission peaks that can be measured simultaneously. Highly fluorescent semiconductor quantum dots with different emission wavelengths (525 nm and 705 nm) were conjugated to anti-E. coli O157 and anti-Salmonella antibodies, respectively. Target bacteria were separated from samples by using specific antibody coated magnetic beads. The bead-cell complexes reacted with QD-antibody conjugates to form bead-cell-QD complexes. Fluorescent microscopic images of QD labeled E. coli and Salmonella cells demonstrated that QD-antibody conjugates could evenly and completely attach to the surface of bacterial cells, indicating that the conjugated QD molecules still retain their effective fluorescence, while the conjugated antibody molecules remain active and are able to recognize their specific target bacteria in a complex mixture. The intensities of fluorescence emission peaks at 525 nm and 705 nm of the final complexes were measured for quantitative detection of E. coli O157:H7 and S. Typhimurium simultaneously. The fluorescence intensity (FI) as a function of cell number (N) was found for Salmonella and E. coli, respectively. The regression models can be expressed as: FI = 60.6 log N- 250.9 with R(2) = 0.97 for S. Typhimurium, and FI = 77.8 log N- 245.2 with R(2) = 0.91 for E. coli O157:H7 in the range of cell numbers from 10(4) to 10(7) cfu ml(-1). The detection limit of this method was 10(4) cfu ml(-1). The detection could be completed within 2 hours. The principle of this method could be extended to detect multiple species of bacteria (3-4 species) simultaneously, depending on the availability of each type of QD-antibody conjugates with a unique emission peak and the antibody coated magnetic beads specific to each species of bacteria.

Genome sequence analyses of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic Escherichia coli (EAHEC).

PubMed

Brzuszkiewicz, Elzbieta; Thürmer, Andrea; Schuldes, Jörg; Leimbach, Andreas; Liesegang, Heiko; Meyer, Frauke-Dorothee; Boelter, Jürgen; Petersen, Heiko; Gottschalk, Gerhard; Daniel, Rolf

2011-12-01

The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum β-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic E scherichia c oli (EAHEC).

Molecular Analysis of Escherichia coli from retail meats (2002-2004) from the United States National Antimicrobial Resistance Monitoring System.

PubMed

Johnson, James R; McCabe, James S; White, David G; Johnston, Brian; Kuskowski, Michael A; McDermott, Patrick

2009-07-15

The origins and virulence potential of meat product-associated Escherichia coli are undefined. Two hundred eighty-seven E. coli isolates (145 resistant and 142 susceptible to trimethoprim-sulfamethoxazole, nalidixic acid, and/or ceftiofur), recovered by the United States National Antimicrobial Monitoring System from retail beef, pork, chicken, and turkey products (from Oregon, Tennessee, Georgia, and Maryland, 2002-2004) underwent polymerase chain reaction testing for phylogenetic groupings and 59 virulence-associated genes. However analyzed, resistant and susceptible isolates differed minimally according to the assessed characteristics. In contrast, the 4 meat types differed greatly for multiple individual traits and aggregate virulence scores. Poultry isolates exhibited virulence genes associated with avian pathogenic E. coli; beef isolates exhibited traits associated with E. coli from diseased cattle. Overall, 20% of isolates qualified as extraintestinal pathogenic E. coli, with poultry isolates exhibiting significantly higher virulence scores than beef and pork isolates (P < .001). Within this systematically collected, geographically distributed sample of recent retail meat isolates, the carriage of extraintestinal pathogenic E. coli virulence genes in antimicrobial-resistant and antimicrobial-susceptible E. coli appeared similar, whereas isolates from different types of meat differed, consistent with on-farm acquisition of resistance within host species-specific E. coli populations. A substantial minority of meat-source E. coli (whether susceptible or resistant) may represent potential human pathogens.

Occurrence of foodborne bacteria in Alberta feedlots.

PubMed

Van Donkersgoed, Joyce; Bohaychuk, Valerie; Besser, Thomas; Song, Xin-Ming; Wagner, Bruce; Hancock, Dale; Renter, David; Dargatz, David

2009-02-01

The occurrence of generic Escherichia coli, E. coli O157, Salmonella, and Campylobacter in cattle manure, beef carcasses, catch basin water, and soils receiving manure application was determined in 21 Alberta feedlots. In cattle manure, generic E. coli (98%, 2069/2100) and Campylobacter (76%, 1590/2100) were frequently detected; E. coli O157 (7%, 143/2100) and Salmonella (1%, 20/2100) were less frequently detected. Samples from beef carcasses in the cooler following Hazard Analysis Critical Control Point interventions yielded only 1 isolate each of generic E. coli and Campylobacter (1/1653) and no Salmonella (0/1653). Catch basin water specimens were positive for generic E. coli in both the spring (62%, 13/21) and the fall (52%, 11/21). Other bacteria were detected only in the spring water specimens, including E. coli O157 (29%, 6/21), Salmonella (5%, 1/21), and Campylobacter (52%, 11/21). Generic E. coli was frequently isolated from soil specimens (30%, 27/88), but E. coli O157 was not found in soil samples obtained in the spring and was only occasionally detected in the fall samples (9%, 3/32). Salmonella were occasionally found in the soil specimens collected in the spring (3%, 2/56), but not in the fall season (0/32). Campylobacter jejuni was frequent in cattle manure (66%, 1070/1623), but rare in carcass and environmental samples. E. coli O157 and Salmonella were rarely detected in cattle or the environment. Generic E. coli and Salmonella were rarely detected on carcasses.

Antimicrobial properties of ternary eutectic aluminum alloys.

PubMed

Hahn, Claudia; Hans, Michael; Hein, Christina; Dennstedt, Anne; Mücklich, Frank; Rettberg, Petra; Hellweg, Christine Elisabeth; Leichert, Lars Ingo; Rensing, Christopher; Moeller, Ralf

2018-06-27

Several Escherichia coli deletion mutants of the Keio collection were selected for analysis to better understand which genes may play a key role in copper or silver homeostasis. Each of the selected E. coli mutants had a deletion of a single gene predicted to encode proteins for homologous recombination or contained functions directly linked to copper or silver transport or transformation. The survival of these strains on pure copper surfaces, stainless steel, and alloys of aluminum, copper and/or silver was investigated. When exposed to pure copper surfaces, E. coli ΔcueO was the most sensitive, whereas E. coli ΔcopA was the most resistant amongst the different strains tested. However, we observed a different trend in sensitivities in E. coli strains upon exposure to alloys of the system Al-Ag-Cu. While minor antimicrobial effects were detected after exposure of E. coli ΔcopA and E. coli ΔrecA to Al-Ag alloys, no effect was detected after exposure to Al-Cu alloys. The release of copper ions and cell-associated copper ion concentrations were determined for E. coli ΔcopA and the wild-type E. coli after exposure to pure copper surfaces. Altogether, compared to binary alloys, ternary eutectic alloys (Al-Ag-Cu) had the highest antimicrobial effect and thus, warrant further investigation.

Odontogenic Differentiation of Human Dental Pulp Stem Cells on Hydrogel Scaffolds Derived from Decellularized Bone Extracellular Matrix and Collagen Type I.

PubMed

Paduano, Francesco; Marrelli, Massimo; White, Lisa J; Shakesheff, Kevin M; Tatullo, Marco

2016-01-01

The aim of this study was to evaluate the level of odontogenic differentiation of dental pulp stem cells (DPSCs) on hydrogel scaffolds derived from bone extracellular matrix (bECM) in comparison to those seeded on collagen I (Col-I), one of the main components of dental pulp ECM. DPSCs isolated from human third molars were characterized for surface marker expression and odontogenic potential prior to seeding into bECM or Col-I hydrogel scaffolds. The cells were then seeded onto bECM and Col-I hydrogel scaffolds and cultured under basal conditions or with odontogenic and growth factor (GF) supplements. DPSCs cultivated on tissue culture polystyrene (TCPS) with and without supplements were used as controls. Gene expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE) was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and mineral deposition was observed by Von Kossa staining. When DPSCs were cultured on bECM hydrogels, the mRNA expression levels of DSPP, DMP-1 and MEPE genes were significantly upregulated with respect to those cultured on Col-I scaffolds or TCPS in the absence of extra odontogenic inducers. In addition, more mineral deposition was observed on bECM hydrogel scaffolds as demonstrated by Von Kossa staining. Moreover, DSPP, DMP-1 and MEPE mRNA expressions of DPSCs cultured on bECM hydrogels were further upregulated by the addition of GFs or osteo/odontogenic medium compared to Col-I treated cells in the same culture conditions. These results demonstrate the potential of the bECM hydrogel scaffolds to stimulate odontogenic differentiation of DPSCs.

Spatio-temporal scan statistics for the detection of outbreaks involving common molecular subtypes: using human cases of Escherichia coli O157:H7 provincial PFGE pattern 8 (National Designation ECXAI.0001) in Alberta as an example.

PubMed

So, H C; Pearl, D L; von Königslöw, T; Louie, M; Chui, L; Svenson, L W

2013-08-01

Molecular typing methods have become a common part of the surveillance of foodborne pathogens. In particular, pulsed-field gel electrophoresis (PFGE) has been used successfully to identify outbreaks of Escherichia coli O157:H7 in humans from a variety of food and environmental sources. However, some PFGE patterns appear commonly in surveillance systems, making it more difficult to distinguish between outbreak and sporadic cases based on molecular data alone. In addition, it is unknown whether these common patterns might have unique epidemiological characteristics reflected in their spatial and temporal distributions. Using E. coli O157:H7 surveillance data from Alberta, collected from 2000 to 2002, we investigated whether E. coli O157:H7 with provincial PFGE pattern 8 (national designation ECXAI.0001) clustered in space, time and space-time relative to other PFGE patterns using the spatial scan statistic. Based on our purely spatial and temporal scans using a Bernoulli model, there did not appear to be strong evidence that isolates of E. coli O157:H7 with provincial PFGE pattern 8 are distributed differently from other PFGE patterns. However, we did identify space-time clusters of isolates with PFGE pattern 8, using a Bernoulli model and a space-time permutation model, which included known outbreaks and potentially unrecognized outbreaks or additional outbreak cases. There were differences between the two models in the space-time clusters identified, which suggests that the use of both models could increase the sensitivity of a quantitative surveillance system for identifying outbreaks involving isolates sharing a common PFGE pattern. © 2012 Blackwell Verlag GmbH.

[Effect of nutritional stress on autophagy in free-living amoeba].

PubMed

Wang, Nan-Ning; Tan, Yu-Zhen; Wang, Hai-Jie

2010-12-30

To investigate the change of autophagy and morphological characteristics of the autophagic structures in free-living amoeba under nutritional stress. Free-living amoebae were incubated on the agaric solid medium which had been covered with Escherichia cdi in control group. In the experiment group, amoebae incubated on the agaric solid medium with E. coli were collected and moved to another solid medium without E. coli and incubated for 12 h. The morphological changes of free-living amoeba in the medium without E. coli were viewed with scanning electron microscope. The changes of autophagy and the structural features of the autophagosome precursors, autophagosomes and autophagolysosomes in amoeba were examined with transmission electron microscope, and the cross-section areas of the autophagic structures and cytoplasm were measured with an image analyzer. The autophagosomes in the organism were labeled with monodansylcadaverine (MDC) staining and quantitated using laser scanning confocal microscope. In the control group, free-living amoebae were all in the form of trophozoite. In the experiment group, trophozoites were induced to transform to cysts gradually. In control group, amoeba was full of fragment of E. coli. There was merely little autophagy with fewer autophagic structures in amoeba. When compared with the control group, the autophagic abilities of amoeba were enhanced significantly, number of autophagic structures increased in the experiment group. In addition, the ratio of the cross-sectional areas of the autophagic structures to that of the cytoplasm of amoeba was greater (P < 0.05 or 0.01). There was fragment of E. coli that was not digested in some of the amoebae. In the circumstance of nutritional stress, amoebic trophozoites were induced to transform to cysts gradually. The autophagic ability of free-living amoeba significantly enhanced.

Antibacterial activity and mechanism of action of ε-poly-L-lysine.

PubMed

Ye, Ruosong; Xu, Hengyi; Wan, Cuixiang; Peng, Shanshan; Wang, Lijun; Xu, Hong; Aguilar, Zoraida P; Xiong, Yonghua; Zeng, Zheling; Wei, Hua

2013-09-13

ε-Poly-L-lysine (ε-PL)(2) is widely used as an antibacterial agent because of its broad antimicrobial spectrum. However, the mechanism of ε-PL against pathogens at the molecular level has not been elucidated. This study investigated the antibacterial activity and mechanism of ε-PL against Escherichia coli O157:H7 CMCC44828. Propidium monoazide-PCR test results indicated that the threshold condition of ε-PL for complete membrane lysis of E. coli O157:H7 was 10 μg/mL (90% mortality for 5 μg/mL). Further verification of the destructive effect of ε-PL on cell structure was performed by atomic force microscopy and transmission electron microscopy. Results showed a positive correlation between reactive oxygen species (ROS)(3) levels and ε-PL concentration in E. coli O157:H7 cells. Moreover, the mortality of E. coli O157:H7 was reduced when antioxidant N-acetylcysteine was added. Results from real-time quantitative PCR (RT-qPCR)(4) indicated that the expression levels of oxidative stress genes sodA and oxyR were up-regulated 4- and 16-fold, respectively, whereas virulence genes eaeA and espA were down-regulated after ε-PL treatment. Expression of DNA damage response (SOS response)(5) regulon genes recA and lexA were also affected by ε-PL. In conclusion, the antibacterial mechanism of ε-PL against E. coli O157:H7 may be attributed to disturbance on membrane integrity, oxidative stress by ROS, and effects on various gene expressions, such as regulation of oxidative stress, SOS response, and changes in virulence. Copyright © 2013. Published by Elsevier Inc.

A quantitative real-time PCR assay for the detection of tetR of Tn10 in Escherichia coli using SYBR Green and the Opticon.

PubMed

Morsczeck, Christian; Langendörfer, Daniel; Schierholz, Jörg Michael

2004-06-30

Bacteria of implant infections are extremely resistant to antibiotics. One reason for this antibiotic resistance are transposons; the well-known transposon Tn10, for example, mediates tetracycline resistance to Escherichia coli. Two genes of Tn10, tetA and tetR, are essential for the mechanism of resistance. These genes encode a drug-specific efflux protein and a tetracycline repressor protein, respectively. Tn10 is also widely used in molecular biology. For example, tTA, a recombinant derivate of tetR, has been utilised for a highly efficient gene regulation system in mammalian cells. We have examined E. coli isolates from implant infections for tetracycline resistance and for the presence of tetR. A real-time PCR assay was developed for detection of tetR with SybrGreen using the Opticon PCR machine of MJ Research. This method offers a quick, sensitive, efficient, and reliable approach to the detection and quantification of genes. Clinical isolates of E. coli were examined successfully for tetracycline resistance and for the presence of tetR. The real-time PCR is effective using a variety of templates including isolated E. coli DNA, pure colonies, or liquid culture sources. Using quantified standard DNA, this assay can accurately detect as few as 15 copies. Moreover, this assay has the ability to quantify the number of tetR genes in the presence of contaminating mammalian DNA. In conclusion, the tetR real-time PCR offers new methods for detection and quantification of tetracycline-resistant bacteria and tTA in transfected cell-lines or transgenic animals.

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

DNA microarray-mediated transcriptional profiling of avian pathogenic Escherichia coli O2 strain E058 during its infection of chicken.

PubMed

Gao, Qingqing; Xia, Le; Liu, Juanhua; Wang, Xiaobo; Gao, Song; Liu, Xiufan

2016-11-01

Avian pathogenic Escherichia coli (APEC) cause typical extraintestinal infections in poultry, including acute fatal septicemia, subacute pericarditis, and airsacculitis. These bacteria most often infect chickens, turkeys, ducks, and other avian species, and therefore pose a significant economic burden on the poultry industry worldwide. Few studies have analyzed the genome-wide transcriptional profile of APEC during infection in vivo. In this study, we examined the genome-wide transcriptional response of APEC O2 strain E058 in an in vivo chicken infection model to better understand the factors necessary for APEC colonization, growth, and survival in vivo. An Affymetrix multigenome DNA microarray, which contains most of the genomic open reading frames of E. coli K-12 strain MG1655, uropathogenic E. coli strain CFT073, and E. coli O157:H7 strain EDL 933, was used to profile the gene expression in APEC E058. We identified the in vivo transcriptional response of APEC E058 bacteria collected directly from the blood of infected chickens. Significant differences in expression levels were detected between the in vivo expression profile and the in vitro expression profile in LB medium. The genes highly expressed during infection were involved in metabolism, iron acquisition or transport, virulence, response to stress, and biological regulation. The reliability of the microarray data was confirmed by performing quantitative real-time PCR on 12 representative genes. Moreover, several significantly upregulated genes, including yjiY, sodA, phoB and spy, were selected to study their role in APEC pathogenesis. The data will help to better understand the mechanisms of APEC pathogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Distinct inflammatory and cytopathic characteristics of Escherichia coli isolates from inflammatory bowel disease patients.

PubMed

Jensen, Stina Rikke; Mirsepasi-Lauridsen, Hengameh Chloé; Thysen, Anna Hammerich; Brynskov, Jørn; Krogfelt, Karen A; Petersen, Andreas Munk; Pedersen, Anders Elm; Brix, Susanne

2015-12-01

Escherichia coli (E. coli) may be implicated in the pathogenesis of inflammatory bowel disease (IBD), as implied from a higher prevalence of mucosa-associated E. coli in the gut of IBD-affected individuals. However, it is unclear whether different non-diarrheagenic E. coli spp. segregate from each other in their ability to promote intestinal inflammation. Herein we compared the inflammation-inducing properties of non-diarrheagenic LF82, 691-04A, E. coli Nissle 1917 (ECN) and eleven new intestinal isolates from different locations in five IBD patients and one healthy control. Viable E. coli were cultured with human monocyte-derived dendritic cells (moDCs) and monolayers of intestinal epithelial cells (IECs), followed by analysis of secreted cytokines, intracellular levels of reactive oxygen species and cellular death. The IBD-associated E. coli LF82 induced the same dose-dependent inflammatory cytokine profile as ECN and ten of the new E. coli isolates displayed as high level IL-12p70, IL-1β, IL-23 and TNF-α from moDCs irrespective of their site of isolation (ileum/colon/faeces), disease origin (diseased/non-diseased) or known virulence factors. Contrarily, 691-04A and one new IBD E. coli isolate induced a different cellular phenotype with enhanced killing of moDCs and IECs, coupled to elevated IL-18. The cytopathic nature of 691-04A and one other IBD E. coli isolate suggests that colonization with specific non-diarrheagenic E. coli could promote intestinal barrier leakage and profound intestinal inflammation, while LF82, ECN and the remaining non-diarrheagenic E. coli isolates hold notorious pro-inflammatory characteristics that can progress inflammation in case of intestinal barrier leakage. Copyright © 2015 Elsevier GmbH. All rights reserved.

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.

Short genome report of cellulose-producing commensal Escherichia coli 1094.

PubMed

Bernal-Bayard, Joaquin; Gomez-Valero, Laura; Wessel, Aimee; Khanna, Varun; Bouchier, Christiane; Ghigo, Jean-Marc

2018-01-01

Bacterial surface colonization and biofilm formation often rely on the production of an extracellular polymeric matrix that mediates cell-cell and cell-surface contacts. In Escherichia coli and many Betaproteobacteria and Gammaproteobacteria cellulose is often the main component of the extracellular matrix. Here we report the complete genome sequence of the cellulose producing strain E. coli 1094 and compare it with five other closely related genomes within E. coli phylogenetic group A. We present a comparative analysis of the regions encoding genes responsible for cellulose biosynthesis and discuss the changes that could have led to the loss of this important adaptive advantage in several E. coli strains. Data deposition: The annotated genome sequence has been deposited at the European Nucleotide Archive under the accession number PRJEB21000.

Rapid detection of Escherichia coli and enterococci in recreational water using an immunomagnetic separation/adenosine triphosphate technique

USGS Publications Warehouse

Bushon, R.N.; Brady, A.M.; Likirdopulos, C.A.; Cireddu, J.V.

2009-01-01

Aims: The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water. Methods and Results: Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis (r = 0.62 and 0.77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci. Conclusions: The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria. Significance and Impact of the Study: The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications. ?? 2009 The Authors.

Identification of microRNAs regulating Escherichia coli F18 infection in Meishan weaned piglets.

PubMed

Wu, Zhengchang; Qin, Weiyun; Wu, Seng; Zhu, Guoqiang; Bao, Wenbin; Wu, Shenglong

2016-11-03

Escherichia coli F18 is mainly responsible for post-weaning diarrhea (PWD) in piglets. The molecular regulation of E. coli F18 resistance in Chinese domestic weaned piglets is still obscure. We used Meishan piglets as model animals to test their susceptibility to E. coli F18. Small RNA duodenal libraries were constructed for E. coli F18-sensitive and -resistant weaned piglets challenged with E. coli F18 and sequenced using Illumina Solexa high-throughput sequencing technology. Sequencing results showed that 3,475,231 and 37,198,259 clean reads were obtained, with 311 known miRNAs differently expressed in resistant and sensitive groups, respectively. Twenty-four miRNAs, including 15 up-regulated and 9 down-regulated, demonstrated more than a 2-fold differential expression between the F18-resistant and -sensitive piglets. Stem-loop RT-qPCR showed that miR-136, miR-196b, miR-499-5p and miR-218-3p significantly expressed in intestinal tissue (p < 0.05). KEGG pathway analysis for target genes revealed that differently expressed miRNAs were involved in infectious diseases, signal transduction and immune system pathways. Interestingly, the expression of miR-218-3p in intestinal tissue had a very significant negative correlation with target DLG5 (P < 0.01). Based on the expression correlation between miRNA and target genes analysis, we speculate that miR-218-3p targeting to DLG5, appears to be very promising candidate for miRNAs involved in response to E. coli F18 infection. The present study provides improved database information on pig miRNAs, better understanding of the genetic basis of E. coli F18 resistance in local Chinese pig breeds and lays a new foundation for identifying novel markers of E. coli F18 resistance. This article was reviewed by Neil R Smalheiser and Weixiong Zhang.

Genomic Analysis of Multidrug-Resistant Escherichia coli from North Carolina Community Hospitals: Ongoing Circulation of CTX-M-Producing ST131-H30Rx and ST131-H30R1 Strains.

PubMed

Kanamori, Hajime; Parobek, Christian M; Juliano, Jonathan J; Johnson, James R; Johnston, Brian D; Johnson, Timothy J; Weber, David J; Rutala, William A; Anderson, Deverick J

2017-08-01

Escherichia coli sequence type 131 (ST131) predominates globally among multidrug-resistant (MDR) E. coli strains. We used whole-genome sequencing (WGS) to investigate 63 MDR E. coli isolates from 7 North Carolina community hospitals (2010 to 2015). Of these, 39 (62%) represented ST131, including 37 (95%) from the ST131- H 30R subclone: 10 (27%) from its H 30R1 subset and 27 (69%) from its H 30Rx subset. ST131 core genomes differed by a median of 15 (range, 0 to 490) single-nucleotide variants (SNVs) overall versus only 7 within H 30R1 (range, 3 to 12 SNVs) and 11 within H 30Rx (range, 0 to 21). The four isolates with identical core genomes were all H 30Rx. Epidemiological and clinical characteristics did not vary significantly by strain type, but many patients with MDR E. coli or H 30Rx infection were critically ill and had poor outcomes. H 30Rx isolates characteristically exhibited fluoroquinolone resistance and CTX-M-15 production, had a high prevalence of trimethoprim-sulfamethoxazole resistance (89%), sul1 (89%), and dfrA17 (85%), and were enriched for specific virulence traits, and all qualified as extraintestinal pathogenic E. coli The high overall prevalence of CTX-M-15 appeared to be possibly attributable to its association with the ST131- H 30Rx subclone and IncF[F2:A1:B-] plasmids. Some phylogenetically clustered non-ST131 MDR E. coli isolates also had distinctive serotypes/ fimH types, fluoroquinolone mutations, CTX-M variants, and IncF types. Thus, WGS analysis of our community hospital source MDR E. coli isolates suggested ongoing circulation and differentiation of E. coli ST131 subclones, with clonal segregation of CTX-M variants, other resistance genes, Inc-type plasmids, and virulence genes. Copyright © 2017 American Society for Microbiology.

Pathogenic Potential, Genetic Diversity, and Population Structure of Escherichia coli Strains Isolated from a Forest-Dominated Watershed (Comox Lake) in British Columbia, Canada

PubMed Central

Mazumder, Asit

2014-01-01

Escherichia coli isolates (n = 658) obtained from drinking water intakes of Comox Lake (2011 to 2013) were screened for the following virulence genes (VGs): stx1 and stx2 (Shiga toxin-producing E. coli [STEC]), eae and the adherence factor (EAF) gene (enteropathogenic E. coli [EPEC]), heat-stable (ST) enterotoxin (variants STh and STp) and heat-labile enterotoxin (LT) genes (enterotoxigenic E. coli [ETEC]), and ipaH (enteroinvasive E. coli [EIEC]). The only genes detected were eae and stx2, which were carried by 37.69% (n = 248) of the isolates. Only eae was harbored by 26.74% (n = 176) of the isolates, representing potential atypical EPEC strains, while only stx2 was detected in 10.33% (n = 68) of the isolates, indicating potential STEC strains. Moreover, four isolates were positive for both the stx2 and eae genes, representing potential EHEC strains. The prevalence of VGs (eae or stx2) was significantly (P < 0.0001) higher in the fall season, and multiple genes (eae plus stx2) were detected only in fall. Repetitive element palindromic PCR (rep-PCR) fingerprint analysis of 658 E. coli isolates identified 335 unique fingerprints, with an overall Shannon diversity (H′) index of 3.653. Diversity varied among seasons over the years, with relatively higher diversity during fall. Multivariate analysis of variance (MANOVA) revealed that the majority of the fingerprints showed a tendency to cluster according to year, season, and month. Taken together, the results indicated that the diversity and population structure of E. coli fluctuate on a temporal scale, reflecting the presence of diverse host sources and their behavior over time in the watershed. Furthermore, the occurrence of potentially pathogenic E. coli strains in the drinking water intakes highlights the risk to human health associated with direct and indirect consumption of untreated surface water. PMID:25548059

Comparative analysis of a modified ecolite method, the colicomplete method, and a most-probable-number method for detecting Escherichia coli in orange juice.

PubMed

Durbin, Gregory W; Salter, Robert

2006-01-01

The Ecolite High Volume Juice (HVJ) presence-absence method for a 10-ml juice sample was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual most-probable-number (MPN) method for analysis of artificially contaminated orange juices. Samples were added to Ecolite-HVJ medium and incubated at 35 degrees C for 24 to 48 h. Fluorescent blue results were positive for glucuronidase- and galactosidase-producing microorganisms, specifically indicative of about 94% of Escherichia coli strains. Four strains of E. coli were added to juices at concentrations of 0.21 to 6.8 CFU/ ml. Mixtures of enteric bacteria (Enterobacter plus Klebsiella, Citrobacter plus Proteus, or Hafnia plus Citrobacter plus Enterobacter) were added to simulate background flora. Three orange juice types were evaluated (n = 10) with and without the addition of the E. coli strains. Ecolite-HVJ produced 90 of 90 (10 of 10 samples of three juice types, each inoculated with three different E. coli strains) positive (blue-fluorescent) results with artificially contaminated E. coli that had MPN concentrations of <0.3 to 9.3 CFU/ml. Ten of 30 E. coli ATCC 11229 samples with MPN concentrations of <0.3 CFU/ml were identified as positive with Ecolite-HVJ. Isolated colonies recovered from positive Ecolite-HVJ samples were confirmed biochemically as E. coli. Thirty (10 samples each of three juice types) negative (not fluorescent) results were obtained for samples contaminated with only enteric bacteria and for uninoculated control samples. A juice manufacturer evaluated citrus juice production with both the Ecolite-HVJ and Colicomplete methods and recorded identical negative results for 95 20-ml samples and identical positive results for 5 20-ml samples artificially contaminated with E. coli. The Ecolite-HVJ method requires no preenrichment and subsequent transfer steps, which makes it a simple and easy method for use by juice producers.

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.

Advances in Molecular Serotyping and Subtyping of Escherichia coli.

PubMed

Fratamico, Pina M; DebRoy, Chitrita; Liu, Yanhong; Needleman, David S; Baranzoni, Gian Marco; Feng, Peter

2016-01-01

Escherichia coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera against the ca. 186 O-antigens and 53 H-flagellar antigens. Phenotypic methods, including bacteriophage typing and O- and H- serotyping for differentiating and characterizing E. coli have been used for many years; however, these methods are generally time consuming and not always accurate. Advances in next generation sequencing technologies have made it possible to develop genetic-based subtyping and molecular serotyping methods for E. coli, which are more discriminatory compared to phenotypic typing methods. Furthermore, whole genome sequencing (WGS) of E. coli is replacing established subtyping methods such as pulsed-field gel electrophoresis, providing a major advancement in the ability to investigate food-borne disease outbreaks and for trace-back to sources. A variety of sequence analysis tools and bioinformatic pipelines are being developed to analyze the vast amount of data generated by WGS and to obtain specific information such as O- and H-group determination and the presence of virulence genes and other genetic markers.

Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach

PubMed Central

Bennett, Bryson D; Yuan, Jie; Kimball, Elizabeth H; Rabinowitz, Joshua D

2009-01-01

This protocol provides a method for quantitating the intracellular concentrations of endogenous metabolites in cultured cells. The cells are grown in stable isotope-labeled media to near-complete isotopic enrichment and then extracted in organic solvent containing unlabeled internal standards in known concentrations. The ratio of endogenous metabolite to internal standard in the extract is determined using mass spectrometry (MS). The product of this ratio and the unlabeled standard amount equals the amount of endogenous metabolite present in the cells. The cellular concentration of the metabolite can then be calculated on the basis of intracellular volume of the extracted cells. The protocol is exemplified using Escherichia coli and primary human fibroblasts fed uniformly with 13C-labeled carbon sources, with detection of 13C-assimilation by liquid chromatography–tandem MS. It enables absolute quantitation of several dozen metabolites over ~1 week of work. PMID:18714298

FDA Escherichia coli Identification (FDA-ECID) Microarray: a Pangenome Molecular Toolbox for Serotyping, Virulence Profiling, Molecular Epidemiology, and Phylogeny

PubMed Central

Patel, Isha R.; Gangiredla, Jayanthi; Lacher, David W.; Mammel, Mark K.; Jackson, Scott A.; Lampel, Keith A.

2016-01-01

ABSTRACT Most Escherichia coli strains are nonpathogenic. However, for clinical diagnosis and food safety analysis, current identification methods for pathogenic E. coli either are time-consuming and/or provide limited information. Here, we utilized a custom DNA microarray with informative genetic features extracted from 368 sequence sets for rapid and high-throughput pathogen identification. The FDA Escherichia coli Identification (FDA-ECID) platform contains three sets of molecularly informative features that together stratify strain identification and relatedness. First, 53 known flagellin alleles, 103 alleles of wzx and wzy, and 5 alleles of wzm provide molecular serotyping utility. Second, 41,932 probe sets representing the pan-genome of E. coli provide strain-level gene content information. Third, approximately 125,000 single nucleotide polymorphisms (SNPs) of available whole-genome sequences (WGS) were distilled to 9,984 SNPs capable of recapitulating the E. coli phylogeny. We analyzed 103 diverse E. coli strains with available WGS data, including those associated with past foodborne illnesses, to determine robustness and accuracy. The array was able to accurately identify the molecular O and H serotypes, potentially correcting serological failures and providing better resolution for H-nontypeable/nonmotile phenotypes. In addition, molecular risk assessment was possible with key virulence marker identifications. Epidemiologically, each strain had a unique comparative genomic fingerprint that was extended to an additional 507 food and clinical isolates. Finally, a 99.7% phylogenetic concordance was established between microarray analysis and WGS using SNP-level data for advanced genome typing. Our study demonstrates FDA-ECID as a powerful tool for epidemiology and molecular risk assessment with the capacity to profile the global landscape and diversity of E. coli. IMPORTANCE This study describes a robust, state-of-the-art platform developed from available whole-genome sequences of E. coli and Shigella spp. by distilling useful signatures for epidemiology and molecular risk assessment into one assay. The FDA-ECID microarray contains features that enable comprehensive molecular serotyping and virulence profiling along with genome-scale genotyping and SNP analysis. Hence, it is a molecular toolbox that stratifies strain identification and pathogenic potential in the contexts of epidemiology and phylogeny. We applied this tool to strains from food, environmental, and clinical sources, resulting in significantly greater phylogenetic and strain-specific resolution than previously reported for available typing methods. PMID:27037122

Application of disease burden to quantitative assessment of health hazards for a decentralized water reuse system.

PubMed

Gao, Tingting; Chen, Rong; Wang, Xiaochang; Ngo, Huu Hao; Li, Yu-You; Zhou, Jinhong; Zhang, Lu

2016-05-01

The aim of this article is to introduce the methodology of disease burden (DB) to quantify the health impact of microbial regrowth during wastewater reuse, using the case study of a decentralized water reuse system in Xi'an Si-yuan University, located in Xi'an, China. Based on field investigation findings, Escherichia coli (E. coli), Salmonella and rotavirus were selected as typical regrowth pathogens causing potential health hazards during the reuse of reclaimed water. Subsequently, major exposure routes including sprinkler irrigation, landscape fountains and toilet flushing were identified. Mathematical models were established to build the relationship between exposure dose and disease burden by calculating the disability adjusted life year (DALY). Results of disease burden for this case study show that DALYs attributed to E. coli were significantly greater than those caused by other pathogens, and DALYs associated with sprinkler irrigation were higher than those originating from other routes. A correlation between exposure dose and disease was obtained by introducing a modified calculation of morbidity, which can extend the assessment endpoint of health risk to disease burden from the conventional infection rate. Copyright © 2016 Elsevier B.V. All rights reserved.

Estimating the Health Risk Associated with the Use of Ecological Sanitation Toilets in Malawi.

PubMed

Kumwenda, Save; Msefula, Chisomo; Kadewa, Wilfred; Ngwira, Bagrey; Morse, Tracy

2017-01-01

Use of Ecological Sanitation (EcoSan) sludge is becoming popular due to increasing price of organic fertilizers in Malawi; however, there is little evidence on the associated risks. Quantitative microbiological risk assessment (QMRA) was done to determine health risks associated with use of EcoSan. Pathogens considered included Escherichia coli (E. coli) , Salmonella, and soil transmitted helminths (STHs). Exponential and Beta Poisson models were used to estimate the risk from helminthic and bacterial pathogens, respectively. Main exposure pathways were through poor storage of sludge, contamination of foods during drying, walking barefoot on the ground contaminated with sludge, pit emptying without protection, and application of sludge in the fields. Estimated annual risk for Ascaris lumbricoides, Taenia, and hookworms was approximately over 5.6 × 10 -1 for both Fossa Alternas (FAs) and Urine Diverting Dry Toilet (UDDTs). Risk from E. coli and Salmonella was 8.9 × 10 -2 and above. The risks were higher than WHO acceptable risk for use of faecal sludge in crops of 10 -4 infections per year. Promoters and users of EcoSan latrines need to consider advocating for strict guidelines to reduce the risk.

Clofibric and ethacrynic acids prevent experimental pyelonephritis by Escherichia coli in mice.

PubMed

Balagué, Claudia E; de Ruiz, Clara Silva; Rey, Rosario; de Duffard, Ana María Evangelista; Nader-Macías, María Elena

2004-11-01

Interfering Escherichia coli attachment to the urinary tract, using P-fimbriation inhibitors, can prevent pyelonephritis. Clofibric and ethacrynic acids are organic compounds structurally related, but with different pharmacological uses. These agents are potentially active in the urinary tract due to its elimination in an unaltered form by the renal route. This study described a pyelonephritogenic E. coli strain, grown in the presence of sub-inhibitory concentrations of clofibric or ethacrynic acids (0.1 and 1 mM, respectively), which exhibits inhibition of P1 erythrocytes agglutination and a drastic decrease in fimbriation, using electron microscopy and quantitative analyses of superficial proteins (decrease to a 17-25% in comparison with the control). In vivo assays were performed using ascending urinary tract infection in mice. The treatment with therapeutic doses of the drugs, administered 2 days before the bacterial challenge and daily until the end of the experiment (22 days), abolished renal infection after 7-10 days of drug exposure. Within this period clofibric acid did not produce adverse effects on the renal parenchyma. However, ethacrynic acid caused pyelitis and tubular cellular desquamation. These results suggested that clofibric acid might be useful in the short-term prophylaxis of urinary tract infection.

Risk of diarrhoea from shallow groundwater contaminated with enteropathogens in the Kathmandu Valley, Nepal.

PubMed

Shrestha, Sadhana; Haramoto, Eiji; Malla, Rabin; Nishida, Kei

2015-03-01

Shallow groundwater is the main water source among many alternatives in the Kathmandu Valley, Nepal, which has a rapidly growing population and intermittent piped water supply. Although human pathogens are detected in groundwater, its health effects are unclear. We estimated risk of diarrhoea from shallow groundwater use using quantitative microbial risk assessment. Escherichia coli, Giardia cyst and Cryptosporidium oocyst levels were analysed in dug and tube wells samples. E. coli concentrations were converted to those of enteropathogenic E. coli (EPEC). Risks from EPEC in dug wells and from Cryptosporidium and Giardia in both dug and tube wells were higher than the acceptable limit (<10⁻⁴ infections/person-year) for both drinking and bathing exposures. Risk from protozoan enteropathogens increased the total risk 10,000 times, indicating that ignoring protozoans could lead to serious risk underestimation. Bathing exposure considerably increased risk, indicating that it is an important pathway. Point-of-use (POU) water treatment decreased the risk six-fold and decreased risk overestimation. Because removal efficiency of POU water treatment has the largest impact on total risk, increasing the coverage and efficiency of POU water treatment could be a practical risk management strategy in the Kathmandu Valley and similar settings.

Improved Method for the Incorporation of Heme Cofactors into Recombinant Proteins Using Escherichia coli Nissle 1917.

PubMed

Fiege, Kerstin; Querebillo, Christine Joy; Hildebrandt, Peter; Frankenberg-Dinkel, Nicole

2018-05-15

Recombinant production of heme proteins in Escherichia coli is often limited by the availability of heme in the host. Therefore, several methods, including the reconstitution of heme proteins after production but prior to purification or the HPEX system, conferring the ability to take up external heme have been developed and used in the past. Here we describe the use of the apathogenic E. coli strain Nissle 1917 (EcN) as a suitable host for the recombinant production of heme proteins. EcN has an advantage over commonly used lab strains in that it is able to take up heme from the environment through the heme receptor ChuA. Expression of several heme proteins from different prokaryotic sources led to high yield and quantitative incorporation of the cofactor when heme was supplied in the growth medium. Comparative UV-vis and resonance Raman measurements revealed that the method employed has significant influence on heme coordination with the EcN system representing the most native situation. Therefore, the use of EcN as a host for recombinant heme protein production represents an inexpensive and straightforward method to facilitate further investigations of structure and function.

Living on the edge: transfer and traffic of E. coli in a confined flow.

PubMed

Figueroa-Morales, Nuris; Leonardo Miño, Gastón; Rivera, Aramis; Caballero, Rogelio; Clément, Eric; Altshuler, Ernesto; Lindner, Anke

2015-08-21

We quantitatively study the transport of E. coli near the walls of confined microfluidic channels, and in more detail along the edges formed by the interception of two perpendicular walls. Our experiments establish the connection between bacterial motion at the flat surface and at the edges and demonstrate the robustness of the upstream motion at the edges. Upstream migration of E. coli at the edges is possible at much larger flow rates compared to motion at the flat surfaces. Interestingly, the speed of bacteria at the edges mainly results from collisions between bacteria moving along this single line. We show that upstream motion not only takes place at the edge but also in an "edge boundary layer" whose size varies with the applied flow rate. We quantify the bacterial fluxes along the bottom walls and the edges and show that they result from both the transport velocity of bacteria and the decrease of surface concentration with increasing flow rate due to erosion processes. We rationalize our findings as a function of local variations in the shear rate in the rectangular channels and hydrodynamic attractive forces between bacteria and walls.

Kinetics of large-scale chromosomal movement during asymmetric cell division in Escherichia coli

PubMed Central

Männik, Jaana; O’Neill, Jordan C.

2017-01-01

Coordination between cell division and chromosome replication is essential for a cell to produce viable progeny. In the commonly accepted view, Escherichia coli realize this coordination via the accurate positioning of its cell division apparatus relative to the nucleoids. However, E. coli lacking proper positioning of its cell division planes can still successfully propagate. Here, we characterize how these cells partition their chromosomes into daughters during such asymmetric divisions. Using quantitative time-lapse imaging, we show that DNA translocase, FtsK, can pump as much as 80% (3.7 Mb) of the chromosome between daughters at an average rate of 1700±800 bp/s. Pauses in DNA translocation are rare, and in no occasions did we observe reversals at experimental time scales of a few minutes. The majority of DNA movement occurs at the latest stages of cell division when the cell division protein ZipA has already dissociated from the septum, and the septum has closed to a narrow channel with a diameter much smaller than the resolution limit of the microscope (~250 nm). Our data suggest that the narrow constriction is necessary for effective translocation of DNA by FtsK. PMID:28234902

Estimating the Health Risk Associated with the Use of Ecological Sanitation Toilets in Malawi

PubMed Central

Msefula, Chisomo; Kadewa, Wilfred; Ngwira, Bagrey; Morse, Tracy

2017-01-01

Use of Ecological Sanitation (EcoSan) sludge is becoming popular due to increasing price of organic fertilizers in Malawi; however, there is little evidence on the associated risks. Quantitative microbiological risk assessment (QMRA) was done to determine health risks associated with use of EcoSan. Pathogens considered included Escherichia coli (E. coli), Salmonella, and soil transmitted helminths (STHs). Exponential and Beta Poisson models were used to estimate the risk from helminthic and bacterial pathogens, respectively. Main exposure pathways were through poor storage of sludge, contamination of foods during drying, walking barefoot on the ground contaminated with sludge, pit emptying without protection, and application of sludge in the fields. Estimated annual risk for Ascaris lumbricoides, Taenia, and hookworms was approximately over 5.6 × 10−1 for both Fossa Alternas (FAs) and Urine Diverting Dry Toilet (UDDTs). Risk from E. coli and Salmonella was 8.9 × 10−2 and above. The risks were higher than WHO acceptable risk for use of faecal sludge in crops of 10−4 infections per year. Promoters and users of EcoSan latrines need to consider advocating for strict guidelines to reduce the risk. PMID:29250122

Development of 23 individual TaqMan® real-time PCR assays for identifying common foodborne pathogens using a single set of amplification conditions.

PubMed

Cremonesi, Paola; Pisani, Laura Francesca; Lecchi, Cristina; Ceciliani, Fabrizio; Martino, Pieranna; Bonastre, Armand Sanchez; Karus, Avo; Balzaretti, Claudia; Castiglioni, Bianca

2014-10-01

Most of the acute intestinal diseases are caused by foodborne pathogens with infants and elderly people being at major risk. The aim of this study was to develop a procedure to simultaneously detect 20 foodborne pathogens in complex alimentary matrices such as milk, cheese and meat. The list of targets include, among the others, Listeria spp., Salmonella spp., Shigella spp., Escherichia coli spp., Campylobacter spp., Clostridium spp. and Staphylococcus aureus. The accuracy of detection was determined by using ATCC strains as positive and negative controls. The achieved sensitivity of each of assays was 1 pg of genomic DNA, which was equivalent to ∼1 cfu. The working ranges of the TaqMan(®) Real-time PCR assays, when used quantitatively on cheese and meat samples inoculated with serial dilution of Listeria spp., Listeria monocytogenes, S. aureus, Salmonella enterica, Shigella boydii, E. coli O157:H7, Bacillus cereus, Campylobacter coli, Yersinia enterocolitica, Enterobacter sakazakii and Pseudomonas aeruginosa was 10(8) cfu/g to 10(4) cfu/g. No matrix interferences were observed. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

A note on the false discovery rate of novel peptides in proteogenomics.

PubMed

Zhang, Kun; Fu, Yan; Zeng, Wen-Feng; He, Kun; Chi, Hao; Liu, Chao; Li, Yan-Chang; Gao, Yuan; Xu, Ping; He, Si-Min

2015-10-15

Proteogenomics has been well accepted as a tool to discover novel genes. In most conventional proteogenomic studies, a global false discovery rate is used to filter out false positives for identifying credible novel peptides. However, it has been found that the actual level of false positives in novel peptides is often out of control and behaves differently for different genomes. To quantitatively model this problem, we theoretically analyze the subgroup false discovery rates of annotated and novel peptides. Our analysis shows that the annotation completeness ratio of a genome is the dominant factor influencing the subgroup FDR of novel peptides. Experimental results on two real datasets of Escherichia coli and Mycobacterium tuberculosis support our conjecture. yfu@amss.ac.cn or xupingghy@gmail.com or smhe@ict.ac.cn Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

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

Sunden, Fanny; Peck, Ariana; Salzman, Julia

Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modesmore » of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.« less

Environmental emission of multiresistant Escherichia coli carrying the colistin resistance gene mcr-1 from German swine farms.

PubMed

Guenther, Sebastian; Falgenhauer, Linda; Semmler, Torsten; Imirzalioglu, Can; Chakraborty, Trinad; Roesler, Uwe; Roschanski, Nicole

2017-05-01

Pigs have been the focus of the worldwide spread of colistin resistance. However, there is little information on the transmission of mcr-1 -containing bacteria into the environment of pig farms. We therefore rescreened environmental Escherichia coli isolates from the surrounding farm areas of three previously mcr-1 -positive swine herds in Germany. Thirty-five mixed bacterial cultures obtained from boot swabs, flies, dog faeces and manure from three pig farms in Germany in 2011-12 were non-selectively recultivated and the presence of the mcr-1 gene was checked by real-time PCR. After separation, single E. coli colonies were subsequently isolated and the presence of mcr-1 was confirmed by PCR and sequencing. In addition, phenotypic antimicrobial resistance screening and WGS followed by phylogenetic analysis and resistance genotyping as well as plasmid typing were performed. Seven mcr-1 -positive E. coli strains originating from environmental boot swabs, dog faeces, stable flies and manure were found. The isolates belonged to five different STs (ST10, ST1011, ST1140, ST5281 and ST342) and harboured extensive additional resistance genes. Comparative plasmid analysis predominantly located mcr-1 on IncX4 plasmids, which are strongly related to a recently described plasmid of human clinical origin (pICBEC72Hmcr). WGS-based analysis of the environmental E. coli isolates of farm surroundings showed clear links to mcr-1 -harbouring E. coli recovered from pig production in Europe as well as from human clinical isolates worldwide, presenting another piece of the puzzle, which further complicates the rapidly evolving epidemiology of plasmid-mediated colistin-resistant E. coli strains. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Escherichia coli and coliforms level of food utensils at food premises in Kuala Pilah, Malaysia

NASA Astrophysics Data System (ADS)

Saipullizan, Siti Nurul Ain; Mutalib, Sahilah Abd.; Sedek, Razalee

2018-04-01

The aim of this study was to evaluate the Escherichia coli and coliforms level of food utensils at sixty food premises in Kuala Pilah, Malaysia. The swab technique was used to investigate the presence of E. coli and coliforms on 180 of food utensils (chopping boards: 60, knives: 60 and dish plates: 60). The results indicated that 180 of food utensils were contaminated with E. coli and coliforms. More than 60% of the coliforms were exceeded the acceptable limit compared to E. coli on the food utensils. The one-way ANOVA analysis demonstrated that the presence of E. coli and coliforms differed significantly among the three types of food utensils (knives, chopping boards, and dish plates). In conclusion, this study revealed that there are hygiene issues exist in every food premises. The food utensils are safe to use during the food preparation if the presence of E. coli and coliforms are not exceed the acceptable limit. Thus, it is recommended that the food handlers in the food premises should improve effective cleaning and sanitizing of food utensils in their daily work.

Enhancement of L-cysteine production by disruption of yciW in Escherichia coli.

PubMed

Kawano, Yusuke; Ohtsu, Iwao; Takumi, Kazuhiro; Tamakoshi, Ai; Nonaka, Gen; Funahashi, Eri; Ihara, Masaki; Takagi, Hiroshi

2015-02-01

Using in silico analysis, the yciW gene of Escherichia coli was identified as a novel L-cysteine regulon that may be regulated by the transcriptional activator CysB for sulfur metabolic genes. We found that overexpression of yciW conferred tolerance to L-cysteine, but disruption of yciW increased L-cysteine production in E. coli. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Enlightened Multiscale Simulation of Biochemical Networks. Core Theory, Validating Experiments, and Implementation in Open Software

DTIC Science & Technology

2006-10-01

organisms that can either be in the lysogenic (latent) or lytic (active) state. If following its infection of E . coli , the λ-phage virus enters the...and unfolded proteins (b) in the heat shock response system . . . . . 31 3 Robust stability of the model of Heat Shock in E - coli ...stochastic reachability analysis, all in the context of two biologically motivated and functionally important systems: the heat shock response in E . coli and

Escherichia coli isolates causing asymptomatic bacteriuria in catheterized and noncatheterized individuals possess similar virulence properties.

PubMed

Watts, Rebecca E; Hancock, Viktoria; Ong, Cheryl-Lynn Y; Vejborg, Rebecca Munk; Mabbett, Amanda N; Totsika, Makrina; Looke, David F; Nimmo, Graeme R; Klemm, Per; Schembri, Mark A

2010-07-01

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli being responsible for >80% of all cases. Asymptomatic bacteriuria (ABU) occurs when bacteria colonize the urinary tract without causing clinical symptoms and can affect both catheterized patients (catheter-associated ABU [CA-ABU]) and noncatheterized patients. Here, we compared the virulence properties of a collection of ABU and CA-ABU nosocomial E. coli isolates in terms of antibiotic resistance, phylogenetic grouping, specific UTI-associated virulence genes, hemagglutination characteristics, and biofilm formation. CA-ABU isolates were similar to ABU isolates with regard to the majority of these characteristics; exceptions were that CA-ABU isolates had a higher prevalence of the polysaccharide capsule marker genes kpsMT II and kpsMT K1, while more ABU strains were capable of mannose-resistant hemagglutination. To examine biofilm growth in detail, we performed a global gene expression analysis with two CA-ABU strains that formed a strong biofilm and that possessed a limited adhesin repertoire. The gene expression profile of the CA-ABU strains during biofilm growth showed considerable overlap with that previously described for the prototype ABU E. coli strain, 83972. This is the first global gene expression analysis of E. coli CA-ABU strains. Overall, our data suggest that nosocomial ABU and CA-ABU E. coli isolates possess similar virulence profiles.

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

PubMed

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

2016-11-01

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

Comparative Genomic Analysis of Globally Dominant ST131 Clone with Other Epidemiologically Successful Extraintestinal Pathogenic Escherichia coli (ExPEC) Lineages.

PubMed

Shaik, Sabiha; Ranjan, Amit; Tiwari, Sumeet K; Hussain, Arif; Nandanwar, Nishant; Kumar, Narender; Jadhav, Savita; Semmler, Torsten; Baddam, Ramani; Islam, Mohammed Aminul; Alam, Munirul; Wieler, Lothar H; Watanabe, Haruo; Ahmed, Niyaz

2017-10-24

Escherichia coli sequence type 131 (ST131), a pandemic clone responsible for the high incidence of extraintestinal pathogenic E. coli (ExPEC) infections, has been known widely for its contribution to the worldwide dissemination of multidrug resistance. Although other ExPEC-associated and extended-spectrum-β-lactamase (ESBL)-producing E. coli clones, such as ST38, ST405, and ST648 have been studied widely, no comparative genomic data with respect to other genotypes exist for ST131. In this study, comparative genomic analysis was performed for 99 ST131 E. coli strains with 40 genomes from three other STs, including ST38 ( n = 12), ST405 ( n = 10), and ST648 ( n = 18), and functional studies were performed on five in-house strains corresponding to the four STs. Phylogenomic analysis results from this study corroborated with the sequence type-specific clonality. Results from the genome-wide resistance profiling confirmed that all strains were inherently multidrug resistant. ST131 genomes showed unique virulence profiles, and analysis of mobile genetic elements and their associated methyltransferases (MTases) has revealed that several of them were missing from the majority of the non-ST131 strains. Despite the fact that non-ST131 strains lacked few essential genes belonging to the serum resistome, the in-house strains representing all four STs demonstrated similar resistance levels to serum antibactericidal activity. Core genome analysis data revealed that non-ST131 strains usually lacked several ST131-defined genomic coordinates, and a significant number of genes were missing from the core of the ST131 genomes. Data from this study reinforce adaptive diversification of E. coli strains belonging to the ST131 lineage and provide new insights into the molecular mechanisms underlying clonal diversification of the ST131 lineage. IMPORTANCE E. coli , particularly the ST131 extraintestinal pathogenic E. coli (ExPEC) lineage, is an important cause of community- and hospital-acquired infections, such as urinary tract infections, surgical site infections, bloodstream infections, and sepsis. The treatment of infections caused by ExPEC has become very challenging due to the emergence of resistance to the first-line as well as the last-resort antibiotics. This study analyzes E. coli ST131 against three other important and globally distributed ExPEC lineages (ST38, ST405, and ST648) that also produced extended-spectrum β-lactamase (ESBL). This is perhaps the first study that employs the high-throughput whole-genome sequence-based approach to compare and study the genomic features of these four ExPEC lineages in relation to their functional properties. Findings from this study highlight the differences in the genomic coordinates of ST131 with respect to the other STs considered here. Results from this comparative genomics study can help in advancing the understanding of ST131 evolution and also offer a framework towards future developments in pathogen identification and targeted therapeutics to prevent diseases caused by this pandemic E. coli ST131 clone. Copyright © 2017 Shaik et al.

Alignment-Free Design of Highly Discriminatory Diagnostic Primer Sets for Escherichia coli O104:H4 Outbreak Strains

PubMed Central

Bielaszewska, Martina; Karch, Helge; Toth, Ian K.

2012-01-01

Background An Escherichia coli O104:H4 outbreak in Germany in summer 2011 caused 53 deaths, over 4000 individual infections across Europe, and considerable economic, social and political impact. This outbreak was the first in a position to exploit rapid, benchtop high-throughput sequencing (HTS) technologies and crowdsourced data analysis early in its investigation, establishing a new paradigm for rapid response to disease threats. We describe a novel strategy for design of diagnostic PCR primers that exploited this rapid draft bacterial genome sequencing to distinguish between E. coli O104:H4 outbreak isolates and other pathogenic E. coli isolates, including the historical hæmolytic uræmic syndrome (HUSEC) E. coli HUSEC041 O104:H4 strain, which possesses the same serotype as the outbreak isolates. Methodology/Principal Findings Primers were designed using a novel alignment-free strategy against eleven draft whole genome assemblies of E. coli O104:H4 German outbreak isolates from the E. coli O104:H4 Genome Analysis Crowd-Sourcing Consortium website, and a negative sequence set containing 69 E. coli chromosome and plasmid sequences from public databases. Validation in vitro against 21 ‘positive’ E. coli O104:H4 outbreak and 32 ‘negative’ non-outbreak EHEC isolates indicated that individual primer sets exhibited 100% sensitivity for outbreak isolates, with false positive rates of between 9% and 22%. A minimal combination of two primers discriminated between outbreak and non-outbreak E. coli isolates with 100% sensitivity and 100% specificity. Conclusions/Significance Draft genomes of isolates of disease outbreak bacteria enable high throughput primer design and enhanced diagnostic performance in comparison to traditional molecular assays. Future outbreak investigations will be able to harness HTS rapidly to generate draft genome sequences and diagnostic primer sets, greatly facilitating epidemiology and clinical diagnostics. We expect that high throughput primer design strategies will enable faster, more precise responses to future disease outbreaks of bacterial origin, and help to mitigate their societal impact. PMID:22496820

Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions

DOE PAGES

Shatsky, Maxim; Dong, Ming; Liu, Haichuan; ...

2016-04-20

Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris. These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification ofmore » endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.« less

Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions

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

Shatsky, Maxim; Dong, Ming; Liu, Haichuan

Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris. These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification ofmore » endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.« less

Analysis of whole genome sequencing for the Escherichia coli O157:H7 typing phages.

PubMed

Cowley, Lauren A; Beckett, Stephen J; Chase-Topping, Margo; Perry, Neil; Dallman, Tim J; Gally, David L; Jenkins, Claire

2015-04-08

Shiga toxin producing Escherichia coli O157 can cause severe bloody diarrhea and haemolytic uraemic syndrome. Phage typing of E. coli O157 facilitates public health surveillance and outbreak investigations, certain phage types are more likely to occupy specific niches and are associated with specific age groups and disease severity. The aim of this study was to analyse the genome sequences of 16 (fourteen T4 and two T7) E. coli O157 typing phages and to determine the genes responsible for the subtle differences in phage type profiles. The typing phages were sequenced using paired-end Illumina sequencing at The Genome Analysis Centre and the Animal Health and Veterinary Laboratories Agency and bioinformatics programs including Velvet, Brig and Easyfig were used to analyse them. A two-way Euclidian cluster analysis highlighted the associations between groups of phage types and typing phages. The analysis showed that the T7 typing phages (9 and 10) differed by only three genes and that the T4 typing phages formed three distinct groups of similar genomic sequences: Group 1 (1, 8, 11, 12 and 15, 16), Group 2 (3, 6, 7 and 13) and Group 3 (2, 4, 5 and 14). The E. coli O157 phage typing scheme exhibited a significantly modular network linked to the genetic similarity of each group showing that these groups are specialised to infect a subset of phage types. Sequencing the typing phage has enabled us to identify the variable genes within each group and to determine how this corresponds to changes in phage type.

Cloning and Molecular Analysis of a Mannitol Operon of Phosphoenolpyruvate-dependent Phosphotransferase (PTS) type From Vibrio cholerae O395

PubMed Central

Kumar, Sanath; Smith, Kenneth P.; Floyd, Jody L.; Varela, Manuel F.

2010-01-01

A putative mannitol operon of the phosphoenolpyruvate phosphotransferase (PTS) type was cloned from Vibrio cholerae O395 and its activity studied in Escherichia coli. The 3.9 kb operon comprising of three genes is organized as mtlADR. Based on the sequence analysis, these were identified as genes encoding a putative mannitol-specific enzyme IICBA (EIIMtl) component (MtlA), a mannitol-1-phosphate dehydrogenase (MtlD) and a mannitol operon repressor (MtlR). The transport of [3H]mannitol by the cloned mannitol operon in E. coli was 13.8±1.4 nmol/min/mg protein. The insertional inactivation of EIIMtl abolished mannitol and sorbitol transport in V. cholerae O395. Comparison of the mannitol utilization apparatus of V. cholerae with those of Gram-negative and Gram positive bacteria suggests highly conserved nature of the system. MtlA and MtlD exhibit 75% similarity with corresponding sequences of E. coli mannitol operon genes, while MtlR has 63% similarity with MtlR of E. coli. The cloning of V. cholerae mannitol utilization system in an E. coli background will help in elucidating the functional properties of this operon. PMID:21184218

Characterization of beta-carotene ketolases, CrtW, from marine bacteria by complementation analysis in Escherichia coli.

PubMed

Choi, Seon-kang; Nishida, Yasuhiro; Matsuda, Satoru; Adachi, Kyoko; Kasai, Hiroaki; Peng, Xue; Komemushi, Sadao; Miki, Wataru; Misawa, Norihiko

2005-01-01

A complementation analysis was performed in Escherichia coli to evaluate the efficiency of beta-carotene ketolases (CrtW) from the marine bacteria Brevundimonas sp. SD212, Paracoccus sp. PC1 (Alcaligenes PC-1), and Paracoccus sp. N81106 (Agrobacterium aurantiacum), for astaxanthin production. Each crtW gene was expressed in Escherichia coli synthesizing zeaxanthin due to the presence of plasmid pACCAR25DeltacrtX. Carotenoids that accumulated in the resulting E. coli transformants were examined by chromatographic and spectroscopic analyses. The transformant carrying the Paracoccus sp. PC1 or N81106 crtW gene accumulated high levels of adonixanthin, which is the final astaxanthin precursor for CrtW, and astaxanthin, while the E. coli transformant with crtW from Brevundimonas sp. SD212 did not accumulate any adonixanthin and produced a high level of astaxanthin. These results show efficient conversion by CrtW of Brevundimonas sp. SD212 from adonixanthin to astaxanthin, which is a new-found characteristic of a bacterial CrtW enzyme. The phylogenetic positions between CrtW of the two genera, Brevundimonas and Paracoccus, are distant, although they fall into alpha-Proteobacteria.

Improvement of constraint-based flux estimation during L-phenylalanine production with Escherichia coli using targeted knock-out mutants.

PubMed

Weiner, Michael; Tröndle, Julia; Albermann, Christoph; Sprenger, Georg A; Weuster-Botz, Dirk

2014-07-01

Fed-batch production of the aromatic amino acid L-phenylalanine was studied with recombinant Escherichia coli strains on a 15 L-scale using glycerol as carbon source. Flux Variability Analysis (FVA) was applied for intracellular flux estimation to obtain an insight into intracellular flux distribution during L-phenylalanine production. Variability analysis revealed great flux uncertainties in the central carbon metabolism, especially concerning malate consumption. Due to these results two recombinant strains were genetically engineered differing in the ability of malate degradation and anaplerotic reactions (E. coli FUS4.11 ΔmaeA pF81kan and E. coli FUS4.11 ΔmaeA ΔmaeB pF81kan). Applying these malic enzyme knock-out mutants in the standardized L-phenylalanine production process resulted in almost identical process performances (e.g., L-phenylalanine concentration, production rate and byproduct formation). This clearly highlighted great redundancies in central metabolism in E. coli. Uncertainties of intracellular flux estimations by constraint-based analyses during fed-batch production of L-phenylalanine were drastically reduced by application of the malic enzyme knock-out mutants. © 2014 Wiley Periodicals, Inc.

Genomic anatomy of Escherichia coli O157:H7 outbreaks

PubMed Central

Eppinger, Mark; Mammel, Mark K.; Leclerc, Joseph E.; Ravel, Jacques; Cebula, Thomas A.

2011-01-01

The rapid emergence of Escherichia coli O157:H7 from an unknown strain in 1982 to the dominant hemorrhagic E. coli serotype in the United States and the cause of widespread outbreaks of human food-borne illness highlights a need to evaluate critically the extent to which genomic plasticity of this important enteric pathogen contributes to its pathogenic potential and its evolution as well as its adaptation in different ecological niches. Aimed at a better understanding of the evolution of the E. coli O157:H7 pathogenome, the present study presents the high-quality sequencing and comparative phylogenomic analysis of a comprehensive panel of 25 E. coli O157:H7 strains associated with three nearly simultaneous food-borne outbreaks of human disease in the United States. Here we present a population genetic analysis of more than 200 related strains recovered from patients, contaminated produce, and zoonotic sources. High-resolution phylogenomic approaches allow the dynamics of pathogenome evolution to be followed at a high level of phylogenetic accuracy and resolution. SNP discovery and study of genome architecture and prophage content identified numerous biomarkers to assess the extent of genetic diversity within a set of clinical and environmental strains. A total of 1,225 SNPs were identified in the present study and are now available for typing of the E. coli O157:H7 lineage. These data should prove useful for the development of a refined phylogenomic framework for forensic, diagnostic, and epidemiological studies to define better risk in response to novel and emerging E. coli O157:H7 resistance and virulence phenotypes. PMID:22135463

Use of multiple-locus variable-number tandem repeat analysis to evaluate Escherichia coli O157 subtype distribution and transmission dynamics following natural exposure on a closed beef feedlot facility.

PubMed

Williams, Michele L; Pearl, David L; Bishop, Katherine E; Lejeune, Jeffrey T

2013-10-01

To better understand the epizootiology of Escherichia coli O157:H7 among cattle, all E. coli O157 isolates recovered on a research feedlot during a single feeding period were characterized by multiple-locus variable-number tandem repeat analysis (MLVA). Three distinct MLVA subtypes (A, B, C), accounting for 24%, 15%, and 64% of total isolates, respectively, were identified. Subtypes A and B were isolated at the initiation of sampling, but their prevalence waned and subtype C, first isolated on the third sampling date, became the predominant subtype on the feedlot. Supershedding events, however, occurred with equal frequency for all three MLVA-types. Using a multilevel logistic regression model, we investigated whether the odds of shedding subtype C relative to subtypes A or B were associated with time, diet, or the presence of a penmate shedding high numbers of subtype C. Only time and exposure to an animal shedding MLVA-type C at 10³ colony-forming units or greater in the pen at the time of sampling were significantly associated with increased shedding of subtype C. High-level shedding of those E. coli O157 subtypes better suited for survival in the environment and/or in the host appear to play a significant role in the development of predominant E. coli O157 subtypes. Supershedding events alone are neither required nor sufficient to drive the epidemiology of specific E. coli O157 subtypes. Additional factors are necessary to direct successful on-farm transmission of E. coli O157.

[Verotoxin-producing Escherichia coli: quantitative model of exposure and risk scenarios in cattle carcasses in Argentina].

PubMed

Olvera, Adib; Signorini, Marcelo; Tarabla, Héctor

2010-06-01

Quantify contamination by verotoxin-producing Escherichia coli associated with hemolytic uremic syndrome (VTEC-HUS) in cattle carcasses and generate estimates of exposure in three likely scenarios. A model was constructed of the frequency and magnitude of VTEC-HUS contamination from primary production to the removal of the carcasses from cold storage, based on the published scientific information, epidemiological data, and information from local experts. The probability distributions that best described each step in the process and scenarios were input to the @Risk program with multiple simulations using Monte Carlo analysis. Pearson s correlation test was used for the sensitivity analysis. The estimated frequency of carcasses with VTEC-HUS was 0.37 (95% CI: 0.26 to 0.58) and the final load of VTEC-HUS was 0.47 log CFU/carcass (95% CI: -2.46 to 3.62). The most closely related variables were the fattening system (r = -0.681) and the theoretical concentration of VTEC-HUS on the cattle's skin (r = 0.702). Vaccinating the animals reduced the frequency of VTEC-HUS in the carcasses by 54.1%, although there were no significant changes in the final VTEC-HUS load. Washing the carcasses reduced the final load by 0.42 log CFU/carcass compared with the baseline model, without any change in the frequency. A 50%-60% increase in the percentage of animals fattened in pens would increase the frequency of carcasses contaminated with VTEC-HUS by 15%-23%. Vaccinating the animals was the most effective scenario for reducing introduction of the bacteria in the beef production chain. Intensifying livestock production will increase the public health risk due to greater exposure to VTEC-HUS.

Screening host proteins required for bacterial adherence after H9N2 virus infection.

PubMed

Ma, Li-Li; Sun, Zhen-Hong; Xu, Yu-Lin; Wang, Shu-Juan; Wang, Hui-Ning; Zhang, Hao; Hu, Li-Ping; Sun, Xiao-Mei; Zhu, Lin; Shang, Hong-Qi; Zhu, Rui-Liang; Wei, Kai

2018-01-01

H9N2 subtype low pathogenic avian influenza virus (LPAIV) is distributed worldwide and causes great economic losses in the poultry industry, especially when complicated with other bacterial infections. Tissue damages caused by virus infection provide an opportunity for bacteria invasion, but this mechanism is not sufficient for low pathogenic strains. Moreover, although H9N2 virus infection was demonstrated to promote bacterial infection in several studies, its mechanism remained unclear. In this study, infection experiments in vivo and in vitro demonstrated that the adhesion of Escherichia coli (E. coli) to host cells significantly increased after H9N2 virus infection, and this increase was not caused by pathological damages. Subsequently, we constructed a late chicken embryo infection model and used proteomics techniques to analyze the expression of proteins associated with bacterial adhesion after H9N2 virus infection. A total of 279 significantly differential expressed proteins were detected through isobaric tags for relative and absolute quantitation (iTRAQ) coupled with nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis. The results of Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that differentially expressed proteins were enriched in host innate immunity; cell proliferation, differentiation, and apoptosis; and pathogenicity-related signaling pathways. Finally, we screened out several proteins, such as TGF-β1, integrins, cortactin, E-cadherin, vinculin, and fibromodulin, which were probably associated with bacterial adhesion. The study analyzed the mechanism of secondary bacterial infection induced by H9N2 virus infection from a novel perspective, which provided theoretical and data support for investigating the synergistic infection mechanism between the H9N2 virus and bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

Potential Role of Gut Microbiota in ALS Pathogenesis and Possible Novel Therapeutic Strategies.

PubMed

Mazzini, Letizia; Mogna, Luca; De Marchi, Fabiola; Amoruso, Angela; Pane, Marco; Aloisio, Irene; Cionci, Nicole Bozzi; Gaggìa, Francesca; Lucenti, Ausiliatrice; Bersano, Enrica; Cantello, Roberto; Di Gioia, Diana; Mogna, Giovanni

2018-05-18

Recent preclinical studies suggest that dysfunction of gastrointestinal tract may play a role in amyotrophic lateral sclerosis (ALS) pathogenesis through a modification of the gut microbiota brain axis. Our study is the first focused on microbiota analysis in ALS patients. Our aim was to study the main human gut microbial groups and the overall microbial diversity in ALS and healthy subjects. Moreover we have examined the influence of a treatment with a specific bacteriotherapy composed of Lactobacillus strains (Lactobacillus fermentum, Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus salivarius) acting on the gastrointestinal barrier. We enrolled 50 ALS patients and 50 healthy controls, matched for sex, age, and origin. Fecal samples were used for total genomic DNA extraction. Enterobacteria, Bifidobacterium spp., Lactobacillus spp., Clostridium sensu stricto, Escherichia coli and yeast were quantified using quantitative polymerase chain reaction approach. Denaturing gradient gel electrophoresis analyses were performed to investigate total eubacteria and yeasts populations. Patients were randomized to double-blind treatment either with microorganisms or placebo for 6 months and monitored for clinical progression and microbiota composition. The comparison between ALS subjects and healthy group revealed a variation in the intestinal microbial composition with a higher abundance of E. coli and enterobacteria and a low abundance of total yeast in patients. Polymerase chain reaction denaturing gradient gel electrophoresis analysis showed a cluster distinction between the bacterial profiles of ALS patients and the healthy subjects. The complexity of the profiles in both cases may indicate that a real dysbiosis status is not evident in the ALS patients although differences between healthy and patients exist. The effects of the progression of the disease and of the bacteriotherapy on the bacterial and yeast populations are currently in progress. Our preliminary results confirm that there is a difference in the microbiota profile in ALS patients.

Distribution and abundance of human-specific Bacteroides and relation to traditional indicators in an urban tropical catchment

PubMed Central

Nshimyimana, J P; Ekklesia, E; Shanahan, P; Chua, L H C; Thompson, J R

2014-01-01

Aims The study goals were to determine the relationship between faecal indicator bacteria (FIB), the HF183 marker and land use, and the phylogenetic diversity of HF183 marker sequences in a tropical urban watershed. Methods and Results Total coliforms, Escherichia coli, and HF183 were quantified in 81 samples categorized as undeveloped, residential and horticultural from the Kranji Reservoir and Catchment in Singapore. Quantitative-PCR for HF183 followed by analysis of variance indicated that horticultural areas had significantly higher geometric means for marker levels (4·3 × 104 HF183-GE 100 ml−1) than nonhorticultural areas (3·07 × 103 HF183-GE 100 ml−1). E. coli and HF183 were moderately correlated in horticultural areas (R = 0·59, P = 0·0077), but not elsewhere in the catchment. Initial upstream surveys of candidate sources revealed elevated HF183 in a wastewater treatment effluent but not in aquaculture ponds. The HF183 marker was cloned, sequenced and determined by phylogenetic analysis to match the original marker description. Conclusion We show that quantification of the HF183 marker is a useful tool for mapping the spatial distribution and potential sources of human sewage contamination in tropical environments such as Singapore. Significance and Impact A major challenge for assessment of water quality in tropical environments is the natural occurrence and nonconservative behaviour of FIB. The HF183 marker has been employed in temperate environments as an alternative indicator for human sewage contamination. Our study supports the use of the HF183 marker as an indicator for human sewage in Singapore and motivates further work to determine HF183 marker levels that correspond to public health risk in tropical environments. PMID:24460587

Transcriptome Analysis of Escherichia coli O157:H7 Exposed to Lysates of Lettuce Leaves ▿

PubMed Central

Kyle, Jennifer L.; Parker, Craig T.; Goudeau, Danielle; Brandl, Maria T.

2010-01-01

Harvesting and processing of leafy greens inherently cause plant tissue damage, creating niches on leaves that human pathogens can exploit. We previously demonstrated that Escherichia coli O157:H7 (EcO157) multiplies more rapidly on shredded leaves than on intact leaves (M. T. Brandl, Appl. Environ. Microbiol. 74:5285-5289, 2008). To investigate how EcO157 cells adapt to physicochemical conditions in injured lettuce tissue, we used microarray-based whole-genome transcriptional profiling to characterize gene expression patterns in EcO157 after 15- and 30-min exposures to romaine lettuce lysates. Multiple carbohydrate transport systems that have a role in the utilization of substrates known to be prevalent in plant cells were activated in EcO157. This indicates the availability to the human pathogen of a variety of carbohydrates released from injured plant cells that may promote its extensive growth in leaf lysates and, thus, in wounded leaf tissue. In addition, microarray analysis revealed the upregulation of numerous genes associated with EcO157 attachment and virulence, with oxidative stress and antimicrobial resistance (including the OxyR and Mar regulons), with detoxification of noxious compounds, and with DNA repair. Upregulation of oxidative stress and antimicrobial resistance genes in EcO157 was confirmed on shredded lettuce by quantitative reverse transcription-PCR. We further demonstrate that this adaptation to stress conditions imparts the pathogen with increased resistance to hydrogen peroxide and calcium hypochlorite. This enhanced resistance to chlorinated sanitizers combined with increased expression of virulence determinants and multiplication at sites of injury on the leaves may help explain the association of processed leafy greens with outbreaks of EcO157. PMID:20061451

Cloning, expression analysis, and antibacterial propertiesof three serum amyloid A in common carp (Cyprinus carpio).

PubMed

Yu, Juhua; Tang, Yongkai; Li, Jianlin; Li, Hongxia; Yu, Fan; Yu, Wenjuan; He, Feng; Fu, Chunjie; Mao, Shuntao

2017-06-01

Three serum amyloid A (SAA) genes were identified from the common carp (Cyprinus carpio) by PCR and RT-PCR. Considering both direction and sequence similarity with mammal's orthologs, they were named CcSAA3a, CcSAA3b and CcSAA1. CcSAA3b and CcSAA1 are adjacent on contig LHQP01017858, suggesting that the prototype of or the simplest SAA multigene family have occurred in common carp. A phylogenetic analysis of the SAAs indicated that the fish SAAs were closer to those of invertebrates and Ornithorhynchus anatinus, a primitive mammal, than to mammalian SAAs. Quantitative real-time RT-PCR results displayed different expression profiles of three CcSAAs. The CcSAA3a was detected in all tested tissues, and was most abundant in the muscle; CcSAA3b was predominately expressed in the intestine and liver, and CcSAA1 in the skin. The expression level of CcSAA3a was higher than that of CcSAA3b and CcSAA1 in most tissues. Stimulation with Aeromonas hydrophila dramatically induced the expression of the three CcSAAs in all examined tissues, especially in the liver. Like Epinephelus coioides SAA, all of three rCcSAA fusion proteins could bind to both Gram-negative bacteria (A. hydrophila and E. coli) and Gram-positive bacterium (S. aureus), playing a role in the identification of bacteria. However, only rCcSAA3a showed significantly anti-A. hydrophila and anti-E. coli in vitro antibacterial activity assays. These results suggested that the three CcSAAs were in functional differentiation and play significant roles in the innate immunity of common carp. Copyright © 2017. Published by Elsevier Ltd.

Spot test analysis of microbial contents during composting of kitchen- and garden biowaste: sampling procedures, bacterial reductions, time-temperature relationships, and their relevance for EU-regulations concerning animal by-products.

PubMed

Bijlsma, P B; de Wit, D H; Duindam, J W; Elsinga, G J; Elsinga, W

2013-01-30

This study was aimed to collect data and develop methodologies to determine if and how Dutch biowaste composting plants can meet the microbiological requirements set out in EU-Regulations (EC) 1774/2002 and (EC) 1069/2009, and to provide the European Food and Safety Authority (EFSA) with data and analysis for evaluation of these regulations. We examined twenty plant locations and four types of composting technologies, all with forced aeration and without an anaerobic digestion phase. Raw biowaste, material after sanitation and compost were sampled by spot test analysis according to a standard protocol, and according to an additional protocol with enhanced hygienic precautions. Samples were analyzed for Escherichia coli, Enterococcaceae and Salmonella content. The latter protocol resulted in improved bacterial reductions after sanitation, whereas in compost Enterococcus levels but not E. coli levels increased substantially with both protocols, due to more thermo-resistant regrowth. Salmonella presence in compost coincided with low temperatures and increased levels of E. coli and Enterococcus, absence of Salmonella was associated with absence of E. coli (74%), but not with absence of Enterococcus (17%). In compost, E. coli and Salmonella showed a comparable time-temperature inactivation pattern. A pilot study with co-composting of biowaste and poultry manure indicated a similar inactivation pattern for ESBL-containing bacteria. We conclude that the abundance of Enterococcus in compost is caused by regrowth and not by (re)contamination, and that E. coli is a more reliable indicator species for the absence/presence of Salmonella in compost. Compliance with current EU-regulations concerning biowaste composting can be shown by spot test analysis at all examined plants, provided that adequate hygienic precautions are taken during sampling. Copyright © 2012 Elsevier Ltd. All rights reserved.

Adhesive force between graphene nanoscale flakes and living biological cells.

PubMed

Al Faouri, Radwan; Henry, Ralph; Biris, Alexandru S; Sleezer, Rob; Salamo, Gregory J

2017-11-01

We report on a measurement technique that quantifies the adhesive force between multi-layers of graphene flakes and the cell wall of live Escherichia coli cells using atomic force microscopy (AFM) in-fluid Peak Force- Quantitative Nanomechanical Mapping mode. To measure the adhesive force, we made use of the negative charge of E. coli cells to allow them to stick to positively charged surfaces, such as glass or silicon, that were covered by poly-L-Lysine. With this approach, cells were held in place for AFM characterization. Both pristine graphene (PG) flakes and functionalized graphene (FG) flakes were put on the E. coli cells and measurements of lateral size, flake thickness, and adhesion were made. Using this approach, the measured values of the adhesive force between multi-layers of graphene flakes (total thickness of 50 nm) and E. coli was determined to be equal or greater than 431 ± 65pN for (PG) and 694 ± 98pN for the (FG). More interestingly, the adhesive force of a graphene flake (thickness 1.3 nm) with the cell is determined to be equal or greater than 38.2 ± 16.4pN for the (PG) and 34.8 ± 15.3pN for the (FG). These interaction values can play an important role in determining and understanding the possible toxicity of graphene flakes. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Comparison of fecal indicators with pathogenic bacteria and rotavirus in groundwater.

PubMed

Ferguson, Andrew S; Layton, Alice C; Mailloux, Brian J; Culligan, Patricia J; Williams, Daniel E; Smartt, Abby E; Sayler, Gary S; Feighery, John; McKay, Larry D; Knappett, Peter S K; Alexandrova, Ekaterina; Arbit, Talia; Emch, Michael; Escamilla, Veronica; Ahmed, Kazi Matin; Alam, Md Jahangir; Streatfield, P Kim; Yunus, Mohammad; van Geen, Alexander

2012-08-01

Groundwater is routinely analyzed for fecal indicators but direct comparisons of fecal indicators to the presence of bacterial and viral pathogens are rare. This study was conducted in rural Bangladesh where the human population density is high, sanitation is poor, and groundwater pumped from shallow tubewells is often contaminated with fecal bacteria. Five indicator microorganisms (E. coli, total coliform, F+RNA coliphage, Bacteroides and human-associated Bacteroides) and various environmental parameters were compared to the direct detection of waterborne pathogens by quantitative PCR in groundwater pumped from 50 tubewells. Rotavirus was detected in groundwater filtrate from the largest proportion of tubewells (40%), followed by Shigella (10%), Vibrio (10%), and pathogenic E. coli (8%). Spearman rank correlations and sensitivity-specificity calculations indicate that some, but not all, combinations of indicators and environmental parameters can predict the presence of pathogens. Culture-dependent fecal indicator bacteria measured on a single date did not predict total bacterial pathogens, but annually averaged monthly measurements of culturable E. coli did improve prediction for total bacterial pathogens. A qPCR-based E. coli assay was the best indicator for the bacterial pathogens. F+RNA coliphage were neither correlated nor sufficiently sensitive towards rotavirus, but were predictive of bacterial pathogens. Since groundwater cannot be excluded as a significant source of diarrheal disease in Bangladesh and neighboring countries with similar characteristics, the need to develop more effective methods for screening tubewells with respect to microbial contamination is necessary. Copyright © 2012 Elsevier B.V. All rights reserved.

Comparison of fecal indicators with pathogenic bacteria and rotavirus in groundwater

PubMed Central

Ferguson, Andrew S.; Layton, Alice C.; Mailloux, Brian J; Culligan, Patricia J.; Williams, Daniel E.; Smartt, Abby E.; Sayler, Gary S.; Feighery, John; McKay, Larry; Knappett, Peter S.K.; Alexandrova, Ekaterina; Arbit, Talia; Emch, Michael; Escamilla, Veronica; Ahmed, Kazi Matin; Alam, Md. Jahangir; Streatfield, P. Kim; Yunus, Mohammad; van Geen, Alexander

2012-01-01

Groundwater is routinely analyzed for fecal indicators but direct comparisons of fecal indicators to the presence of bacterial and viral pathogens are rare. This study was conducted in rural Bangladesh where the human population density is high, sanitation is poor, and groundwater pumped from shallow tubewells is often contaminated with fecal bacteria. Five indicator microorganisms (E. coli, total coliform, F+RNA coliphage, Bacteroides and human-associated Bacteroides) and various environmental parameters were compared to the direct detection of waterborne pathogens by quantitative PCR in groundwater pumped from 50 tubewells. Rotavirus was detected in groundwater filtrate from the largest proportion of tubewells (40%), followed by Shigella (10%), Vibrio (10%), and pathogenic E. coli (8%). Spearman rank correlations and sensitivity-specificity calculations indicate that some, but not all, combinations of indicators and environmental parameters can predict the presence of pathogens. Culture-dependent fecal indicator bacteria measured on a single date did not predict total bacterial pathogens, but annually averaged monthly measurements of culturable E. coli did improve prediction for total bacterial pathogens. A qPCR-based E. coli assay was the best indicator for the bacterial pathogens. F+RNA coliphage were neither correlated nor sufficiently sensitive towards rotavirus, but were predictive of bacterial pathogens. Since groundwater cannot be excluded as a significant source of diarrheal disease in Bangladesh and neighboring countries with similar characteristics, the need to develop more effective methods for screening tubewells with respect to microbial contamination is necessary. PMID:22705866

Survival potential of Escherichia coli and Enterococci in subtropical beach sand: implications for water quality managers.

PubMed

Hartz, A; Cuvelier, M; Nowosielski, K; Bonilla, T D; Green, M; Esiobu, N; McCorquodale, D S; Rogerson, A

2008-01-01

Fecal bacteria have traditionally been used as indicator organisms to monitor the quality of recreational waters. Recent work has questioned the robustness of traditional indicators, particularly at seawater bathing beaches. For example, a study of Florida beaches found unexpectedly high abundances of Escherichia coli, fecal coliforms, and enterococci in beach sand. The aim of the present study was to explain these abundances by assessing the survival of E. coli and enterococci in beach sand relative to seawater. We used a combination of quantitative laboratory mesocosm experiments and field observations. Results suggested that E. coli and enterococci exhibited increased survivability and growth in sand relative to seawater. Because fecal bacteria are capable of replicating in sand, at least under controlled laboratory conditions, the results suggest that sand may be an important reservoir of metabolically active fecal organisms. Experiments with "natural" mesocosms (i.e., unsterilized sand or water rich in micropredators and native bacteria) failed to show the same increases in fecal indicators as was found in sterile sand. It is postulated that this was due to predation and competition with indigenous bacteria in these "natural" systems. Nonetheless, high populations of indicators were maintained and recovered from sand over the duration of the experiment as opposed to the die-off noted in water. Indicator bacteria may wash out of sand into shoreline waters during weather and tidal events, thereby decreasing the effectiveness of these indicators as predictors of health risk and complicating the interpretations for water quality managers.

Draft genome sequence of non-shiga toxin-producing Escherichia coli O157 NCCP15738.

PubMed

Kwon, Taesoo; Kim, Jung-Beom; Bak, Young-Seok; Yu, Young-Bin; Kwon, Ki Sung; Kim, Won; Cho, Seung-Hak

2016-01-01

The non-shiga toxin-producing Escherichia coli (non-STEC) O157 is a pathogenic strain that cause diarrhea but does not cause hemolytic-uremic syndrome, or hemorrhagic colitis. Here, we present the 5-Mb draft genome sequence of non-STEC O157 NCCP15738, which was isolated from the feces of a Korean patient with diarrhea, and describe its features and the structural basis for its genome evolution. A total of 565-Mbp paired-end reads were generated using the Illumina-HiSeq 2000 platform. The reads were assembled into 135 scaffolds throughout the de novo assembly. The assembled genome size of NCCP15738 was 5,005,278 bp with an N50 value of 142,450 bp and 50.65 % G+C content. Using Rapid Annotation using Subsystem Technology analysis, we predicted 4780 ORFs and 31 RNA genes. The evolutionary tree was inferred from multiple sequence alignment of 45 E. coli species. The most closely related neighbor of NCCP15738 indicated by whole-genome phylogeny was E. coli UMNK88, but that indicated by multilocus sequence analysis was E. coli DH1(ME8569). A comparison between the NCCP15738 genome and those of reference strains, E. coli K-12 substr. MG1655 and EHEC O157:H7 EDL933 by bioinformatics analyses revealed unique genes in NCCP15738 associated with lysis protein S, two-component signal transduction system, conjugation, the flagellum, nucleotide-binding proteins, and metal-ion binding proteins. Notably, NCCP15738 has a dual flagella system like that in Vibrio parahaemolyticus, Aeromonas spp., and Rhodospirillum centenum. The draft genome sequence and the results of bioinformatics analysis of NCCP15738 provide the basis for understanding the genomic evolution of this strain.

Transfer of Herb-Resistance Plasmid From Escherichia coli to Staphylococcus aureus Residing in the Human Urinary Tract

PubMed Central

Tong, Yan Qing; Xin, Bing; Zhu, Li

2014-01-01

Background: Plasmid transfer among bacteria provides a means for dissemination of resistance. Plasmid Analysis has made it possible to track plasmids that induce resistance in bacterial population. Objectives: To screen the presence of herb-resistance plasmid in Escherichia coli strains and determine the transferability of this resistance plasmid directly from E. coli to the Gram-positive, Staphylococcus aureus. Materials and Methods: The donor strain E. coli CP9 and recipient strain S. aureus RN450RF were isolated from UTI patients. E. coli CP9 was highly resistant to herbal concoction. Isolates of S. aureus RN450RF were fully susceptible. Total plasmid DNA was prepared and transferred into E. coli DH5α. Transconjugants were selected on agar plates containing serial dilutions of herbal concoction. Resistance plasmid was transferred to susceptible S. aureus RN450RFin triple replicas. The mating experiments were repeated twice. Results: The identified 45 kb herb-resistance plasmid could be transferred from E. coli CP9 isolates to E. coli DH5α. As a consequence E. coli DH5α transconjugant MIC increased from 0.0125 g/mL to 0.25 g/mL. The plasmid was easily transferred from E. coli CP9 strain to S. aureus RN450RF with a mean transfer rate of 1×10-2 transconjugants/recipient. The E. coli donor and the S. aureus RN450RF transconjugant contained a plasmid of the same size, which was absent in the recipient before mating. Susceptibility testing showed that the S. aureus RN450RF transconjugant was resistant to herbal concoction. Conclusions: E. coli herb-resistance plasmid can replicate and be expressed in S. aureus. PMID:25147679

Phylogenetic grouping and pathotypic comparison of urine and fecal Escherichia coli isolates from children with urinary tract infection.

PubMed

Navidinia, Masoumeh; Peerayeh, Shahin Najar; Fallah, Fatemeh; Bakhshi, Bita; Sajadinia, Raheleh Sadat

2014-01-01

The aim of this study was to investigate the phylogenetic background and to assess hlyD (involved in the secretion of haemolysin A) and intI1 (encoding a class 1 integrase) in Escherichia coli isolates derived from urinary and fecal specimens. A total of 200 E. coli isolates was collected from patients presenting with urinary tract infection (UTI) during September 2009 to September 2010 and screened for hlyD and intI1 genes by polymerase chain reaction (PCR). Phylogenetic analysis showed that E. coli is composed of four main phylogenetic groups (A, B1, B2 and D) and that uropathogenic E. coli (UPEC) isolates mainly belong to groups B2 (54%) and D (34%) whereas group A (44%) and D (26%) are predominant among commensal E. coli isolates. In this study, hlyD was present in 26% of UPEC and 2% of commensal E. coli isolates. However, hemolytic activity was detected for 42% of UPEC and 6% of commensal E. coli isolates (p < 0.05). intI1 gene was more frequently expressed in UPEC (24%) in comparison with commensal E. coli isolates (12%). Resistance to aztreonam, co-trimoxazole and cefpodoxime were frequently found among UPEC isolates whereas commensal E. coli isolates were commonly resistant to co-trimoxazole, nalidixic acid and cefotaxime. Concluding, a considerable difference between UPEC and commensal E. coli isolates was observed regarding their phylogenetic groups, presence of class 1 integron and hlyD gene, hemolysin activity and resistance pattern. The detection of class 1 integrons and hlyD gene was higher among UPEC compared with commensal E. coli isolates. These findings may contribute for a better understanding of the factors involved in the pathogenesis of UPEC.

Rapid and reliable discrimination between Shigella species and Escherichia coli using MALDI-TOF mass spectrometry.

PubMed

Paauw, Armand; Jonker, Debby; Roeselers, Guus; Heng, Jonathan M E; Mars-Groenendijk, Roos H; Trip, Hein; Molhoek, E Margo; Jansen, Hugo-Jan; van der Plas, Jan; de Jong, Ad L; Majchrzykiewicz-Koehorst, Joanna A; Speksnijder, Arjen G C L

2015-01-01

E. coli-Shigella species are a cryptic group of bacteria in which the Shigella species are distributed within the phylogenetic tree of E. coli. The nomenclature is historically based and the discrimination of these genera developed as a result of the epidemiological need to identify the cause of shigellosis, a severe disease caused by Shigella species. For these reasons, this incorrect classification of shigellae persists to date, and the ability to rapidly characterize E. coli and Shigella species remains highly desirable. Until recently, existing matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) assays used to identify bacteria could not discriminate between E. coli and Shigella species. Here we present a rapid classification method for the E. coli-Shigella phylogroup based on MALDI-TOF MS which is supported by genetic analysis. E. coli and Shigella isolates were collected and genetically characterized by MLVA. A custom reference library for MALDI-TOF MS that represents the genetic diversity of E. coli and Shigella strains was developed. Characterization of E. coli and Shigella species is based on an approach with Biotyper software. Using this reference library it was possible to distinguish between Shigella species and E. coli. Of the 180 isolates tested, 94.4% were correctly classified as E. coli or shigellae. The results of four (2.2%) isolates could not be interpreted and six (3.3%) isolates were classified incorrectly. The custom library extends the existing MALDI-TOF MS method for species determination by enabling rapid and accurate discrimination between Shigella species and E. coli. Copyright © 2015 Elsevier GmbH. All rights reserved.

Solar and temporal effects on Escherichia coli concentration at a Lake Michigan swimming beach

USGS Publications Warehouse

Whitman, Richard L.; Nevers, Meredith B.; Korinek, Ginger C.; Byappanahalli, Muruleedhara N.

2004-01-01

Studies on solar inactivation of Escherichia coli in freshwater and in situ have been limited. At 63rd St. Beach, Chicago, Ill., factors influencing the daily periodicity of culturable E. coli, particularly insolation, were examined. Water samples for E. coli analysis were collected twice daily between April and September 2000 three times a week along five transects in two depths of water. Hydrometeorological conditions were continuously logged: UV radiation, total insolation, wind speed and direction, wave height, and relative lake level. On 10 days, transects were sampled hourly from 0700 to 1500 h. The effect of sunlight on E. coliinactivation was evaluated with dark and transparent in situ mesocosms and ambient lake water. For the study, the number of E. coli samples collected (n) was 2,676. During sunny days, E. coli counts decreased exponentially with day length and exposure to insolation, but on cloudy days, E. coli inactivation was diminished; the E. coli decay rate was strongly influenced by initial concentration. In situ experiments confirmed that insolation primarily inactivated E. coli; UV radiation only marginally affected E. coliconcentration. The relationship between insolation and E. coli density is complicated by relative lake level, wave height, and turbidity, all of which are often products of wind vector. Continuous importation and nighttime replenishment of E. coli were evident. These findings (i) suggest that solar inactivation is an important mechanism for natural reduction of indicator bacteria in large freshwater bodies and (ii) have implications for management strategies of nontidal waters and the use of E. coli as an indicator organism.

Evaluation of Animal Genetic and Physiological Factors That Affect the Prevalence of Escherichia coli O157 in Cattle

PubMed Central

Jeon, Soo Jin; Elzo, Mauricio; DiLorenzo, Nicolas; Lamb, G. Cliff; Jeong, Kwang Cheol

2013-01-01

Controlling the prevalence of Escherichia coli O157 in cattle at the pre-harvest level is critical to reduce outbreaks of this pathogen in humans. Multilayers of factors including the environmental and bacterial factors modulate the colonization and persistence of E. coli O157 in cattle that serve as a reservoir of this pathogen. Here, we report animal factors contributing to the prevalence of E. coli O157 in cattle. We observe the lowest number of E. coli O157 in Brahman breed when compared with other crosses in an Angus-Brahman multibreed herd, and bulls excrete more E. coli O157 than steers in the pens where cattle were housed together. The presence of super-shedders, cattle excreting >105 CFU/rectal anal swab, increases the concentration of E. coli O157 in the pens; thereby super-shedders enhance transmission of this pathogen among cattle. Molecular subtyping analysis reveal only one subtype of E. coli O157 in the multibreed herd, indicating the variance in the levels of E. coli O157 in cattle is influenced by animal factors. Furthermore, strain tracking after relocation of the cattle to a commercial feedlot reveals farm-to-farm transmission of E. coli O157, likely via super-shedders. Our results reveal high risk factors in the prevalence of E. coli O157 in cattle whereby animal genetic and physiological factors influence whether this pathogen can persist in cattle at high concentration, providing insights to intervene this pathogen at the pre-harvest level. PMID:23405204

A systematic review and meta-analysis of the epidemiology of pathogenic Escherichia coli of calves and the role of calves as reservoirs for human pathogenic E. coli.

PubMed

Kolenda, Rafał; Burdukiewicz, Michał; Schierack, Peter

2015-01-01

Escherichia coli bacteria are the most common causes of diarrhea and septicemia in calves. Moreover, calves form a major reservoir for transmission of pathogenic E. coli to humans. Systematic reviews and meta-analyses of publications on E. coli as calf pathogens and the role of calves as reservoir have not been done so far. We reviewed studies between 1951 and 2013 reporting the presence of virulence associated factors (VAFs) in calf E. coli and extracted the following information: year(s) and country of sampling, animal number, health status, isolate number, VAF prevalence, serotypes, diagnostic methods, and biological assays. The prevalence of VAFs or E. coli pathotypes was compared between healthy and diarrheic animals and was analyzed for time courses. Together, 106 papers with 25,982 E. coli isolates from 27 countries tested for VAFs were included. F5, F17, and F41 fimbriae and heat-stable enterotoxin (ST) - VAFs of enterotoxigenic E. coli (ETEC) were significantly associated with calf diarrhea. On the contrary, ETEC VAF F4 fimbriae and heat-labile enterotoxin as well as enteropathogenic (EPEC), Shiga toxin-producing (STEC), and enterohemorrhagic E. coli (EHEC) were not associated with diarrhea. The prevalence increased overtime for ST-positive isolates, but decreased for F5- and STEC-positive isolates. Our study provides useful information about the history of scientific investigations performed in this domain so far, and helps to define etiological agents of calf disease, and to evaluate calves as reservoir hosts for human pathogenic E. coli.

Persistence of culturable Escherichia coli fecal contaminants in dairy alpine grassland soils.

PubMed

Texier, Stéphanie; Prigent-Combaret, Claire; Gourdon, Marie Hélène; Poirier, Marie Andrée; Faivre, Pierre; Dorioz, Jean Marcel; Poulenard, Jérome; Jocteur-Monrozier, Lucile; Moënne-Loccoz, Yvan; Trevisan, Dominique

2008-01-01

Our knowledge of Escherichia coli (E. coli) ecology in the field is very limited in the case of dairy alpine grassland soils. Here, our objective was to monitor field survival of E. coli in cow pats and underlying soils in four different alpine pasture units, and to determine whether the soil could constitute an environmental reservoir. E. coli was enumerated by MPN using a selective medium. E. coli survived well in cow pats (10(7) to 10(8) cells g(-1) dry pat), but cow pats disappeared within about 2 mo. In each pasture unit, constant levels of E. coli (10(3) to 10(4) cells g(-1) dry soil) were recovered from all topsoil (0-5 cm) samples regardless of the sampling date, that is, under the snow cover, immediately after snow melting, or during the pasture season (during and after the decomposition of pats). In deeper soil layers below the root zone (5-25 cm), E. coli persistence varied according to soil type, with higher numbers recovered in poorly-drained soils (10(3) to 10(4) cells g(-1) dry soil) than in well-drained soils (< 10(2) cells g(-1) dry soil). A preliminary analysis of 38 partial uidA sequences of E. coli from pat and soils highlighted a cluster containing sequences only found in this work. Overall, this study raises the possibility that fecal E. coli could have formed a naturalized (sub)population, which is now part of the indigenous soil community of alpine pasture grasslands, the soil thus representing an environmental reservoir of E. coli.

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

Reconstitution of active mycobacterial binuclear iron monooxygenase complex in Escherichia coli.

PubMed

Furuya, Toshiki; Hayashi, Mika; Kino, Kuniki

2013-10-01

Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

Herd-level risk factors associated with the presence of Phage type 21/28 E. coli O157 on Scottish cattle farms

PubMed Central

Halliday, Jo EB; Chase-Topping, Margo E; Pearce, Michael C; McKendrick, Iain J; Allison, Lesley; Fenlon, Dave; Low, Chris; Mellor, Dominic J; Gunn, George J; Woolhouse, Mark EJ

2006-01-01

Background E. coli O157 is a bacterial pathogen that is shed by cattle and can cause severe disease in humans. Phage type (PT) 21/28 is a subtype of E. coli O157 that is found across Scotland and is associated with particularly severe human morbidity. Methods A cross-sectional survey of Scottish cattle farms was conducted in the period Feb 2002-Feb 2004 to determine the prevalence of E. coli O157 in cattle herds. Data from 88 farms on which E. coli O157 was present were analysed using generalised linear mixed models to identify risk factors for the presence of PT 21/28 specifically. Results The analysis identified private water supply, and northerly farm location as risk factors for PT 21/28 presence. There was a significant association between the presence of PT 21/28 and an increased number of E. coli O157 positive pat samples from a farm, and PT 21/28 was significantly associated with larger E. coli O157 counts than non-PT 21/28 E. coli O157. Conclusion PT 21/28 has significant risk factors that distinguish it from other phage types of E. coli O157. This finding has implications for the control of E. coli O157 as a whole and suggests that control could be tailored to target the locally dominant PT. PMID:17140453

Antimicrobial activity of gamma-thionin-like soybean SE60 in E. coli and tobacco plants.

PubMed

Choi, Yeonhee; Choi, Yang Do; Lee, Jong Seob

2008-10-17

The SE60, a low molecular weight, sulfur-rich protein in soybean, is known to be homologous to wheat gamma-purothionin. To elucidate the functional role of SE60, we expressed SE60 cDNA in Escherichia coli and in tobacco plants. A single protein band was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) after anti-FLAG affinity purification of the protein from transformed E. coli. While the control E. coli cells harboring pFLAG-1 showed standard growth with Isopropyl beta-d-1-thiogalactopyranoside (IPTG) induction, E. coli cells expressing the SE60 fusion protein did not grow at all, suggesting that SE60 has toxic effects on E. coli growth. Genomic integration and the expression of transgene in the transgenic tobacco plants were confirmed by Southern and Northern blot analysis, respectively. The transgenic plants demonstrated enhanced resistance against the pathogen Pseudomonas syringae. Taken together, these results strongly suggest that SE60 has antimicrobial activity and play a role in the defense mechanism in soybean plants.

Rapid flow cytometry analysis of antimicrobial properties of nettle powder and cranberry powder

NASA Astrophysics Data System (ADS)

Hattuniemi, Maarit; Korhonen, Johanna; Jaakkola, Mari; Räty, Jarkko; Virtanen, Vesa

2010-11-01

Both nettle (Urtica dioica) and cranberry (Vaccinium oxycoccus) are widely known to have good influence on health. The aim of this study was to investigate antimicrobial properties of nettle powder and cranberry powder against Escherichia coli (E. coli) and monitor the growth of the bacteria by a rapid flow cytometry (FCM) method. For FCM measurements samples were stained with fluorescent dyes. The inhibitory effects of plant material on growth of E. coli were estimated by comparing the results of control sample (E. coli) to E. coli samples with plant material. FCM offers both a brilliant tool to investigate the kinetics of the growth of bacterium, since subsamples can be taken from the same liquid medium during the growing period and with fluorescent dyes a rapid method to investigate viability of the bacterium.

A CASE STUDY OF NONPOINT SOURCES BACTERIAL CONTRIBUTION TO RURAL SURFACE WATER

EPA Science Inventory

The presentation will address several bacterial issues affecting the Turkey Creek (TC) watershed, in north central Ok. Our results from seasonal stream Escherichia coli (E. coli) analysis, bacterial source tracking, and antibiotic resistance will be shared and discussed in relat...

[Multilocus Sequence Typing analysis of human Campylobacter coli in Granada (Spain)].

PubMed

Carrillo-Ávila, J A; Sorlózano-Puerto, A; Pérez-Ruiz, M; Gutiérrez-Fernández, J

2016-12-01

Different subtypes of Campylobacter spp. have been associated with diarrhoea and a Multilocus Sequence Typing (MLST) method has been performed for subtyping. In the present work, MLST was used to analyse the genetic diversity of eight strains of Campylobacter coli. Nineteen genetic markers were amplified for MLST analysis: AnsB, DmsA, ggt, Cj1585c, CJJ81176-1367/1371, Tlp7, cj1321-cj1326, fucP, cj0178, cj0755/cfrA, ceuE, pldA, cstII, cstIII. After comparing the obtained sequences with the Campylobacter MLST database, the allele numbers, sequence types (STs) and clonal complexes (CCs) were assigned. The 8 C. coli isolates yielded 4 different STs belonging to 2 CCs. Seven isolates belong to ST-828 clonal complex and only one isolate belong to ST-21. Two samples came from the same patient, but were isolated in two different periods of time. MLST can be useful for taxonomic characterization of C. coli isolates.

Hazard analysis of Escherichia coli O157:H7 contamination during beef slaughtering in Calvados, France.

PubMed

Guyon, R; Dorey, F; Malas, J P; Leclercq, A

2001-09-01

To identify hazard points and critical points during beef slaughtering, which is a necessary first step toward developing a hazard analysis and critical control point system to control meat contamination by Escherichia coli O157:H7, samples (n = 192) from surfaces, work tops, worker's hands, and beef carcasses were collected from a slaughterhouse in Calvados, France. Five strains of E. coli O157:H7 were isolated from a footbridge and a worker's apron at the preevisceration post and from a worker's hand at the defatting post. Three isolates carried stx2c, eae, and EHEC-hlyA genes and showed similar molecular types by random amplified polymorphic DNA, polymerase chain reaction IS3, and XbaI pulsed-field gel electrophoresis. Thus, this study has shown that preevisceration and defatting post and associated worker's materials are critical points for carcasses contamination by E. coli O157:H7 during beef slaughtering.

Escherichia coli K-12: a cooperatively developed annotation snapshot—2005

PubMed Central

Riley, Monica; Abe, Takashi; Arnaud, Martha B.; Berlyn, Mary K.B.; Blattner, Frederick R.; Chaudhuri, Roy R.; Glasner, Jeremy D.; Horiuchi, Takashi; Keseler, Ingrid M.; Kosuge, Takehide; Mori, Hirotada; Perna, Nicole T.; Plunkett, Guy; Rudd, Kenneth E.; Serres, Margrethe H.; Thomas, Gavin H.; Thomson, Nicholas R.; Wishart, David; Wanner, Barry L.

2006-01-01

The goal of this group project has been to coordinate and bring up-to-date information on all genes of Escherichia coli K-12. Annotation of the genome of an organism entails identification of genes, the boundaries of genes in terms of precise start and end sites, and description of the gene products. Known and predicted functions were assigned to each gene product on the basis of experimental evidence or sequence analysis. Since both kinds of evidence are constantly expanding, no annotation is complete at any moment in time. This is a snapshot analysis based on the most recent genome sequences of two E.coli K-12 bacteria. An accurate and up-to-date description of E.coli K-12 genes is of particular importance to the scientific community because experimentally determined properties of its gene products provide fundamental information for annotation of innumerable genes of other organisms. Availability of the complete genome sequence of two K-12 strains allows comparison of their genotypes and mutant status of alleles. PMID:16397293

An enzootic outbreak of acute disease associated with pathogenic E. coli in Adler monkey colony.

PubMed

Lapin, Boris A; Yakovleva, Lelita A; Dzhikidze, Eteri K; Gvozdik, Tatiana E; Agumava, Aslan A; Stasilevich, Zinaida K; Danilova, Irina G

2015-12-01

In spring 2009 in Adler colony of the Institute of Medical Primatology, a large enzootic outbreak of acute intestine infection associated with pathogenic E. coli occurred and caused 5% mortality of population (209 animals). The epidemiological analysis, bacteriological investigation, postmortem examination, histological analysis, and PCR were used to identify the infectious agent. Marked hemorrhagic diathesis, lethargy, dehydration, diarrhea with blood, wasting, and sometimes dystrophic changes in articular cartilages were noted. Morphologically, hemorrhagic enterocolitis and massive hemorrhages were found. PCR investigation of bacteriologically isolated E. coli characterized it as enteropathogenic and enteroinvasive E. coli. The outbreak in Adler colony slightly differed from similar outbreak in Florida in 2014 by more marked hemorrhagic diathesis and articular changes in some monkeys caused by polyavitaminosis developed in the course of infection. Sensitive to infection were M. mulatta, M. fascicularis, Cercopithecus aethiops, P. hamadryas and anubis, and Cebus capucinus. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.