The DNA Sequence of the Escherichia coli O22 O-Antigen Gene Cluster and Detection of Pathogenic Strains Belonging to E. coli Serogroups O22 and O91 by Multiplex PCR Assays Targeting Virulence Genes and Genes in the Respective O-Antigen Gene Clusters

Multiplex polymerase chain reaction (PCR) assays were developed for detection of pathogenic strains belonging to Escherichia coli serogroups O22 and O91. The O-antigen gene cluster of E. coli O22 was sequenced to identify genes that could be employed as targets for serogroup-specific PCR assays. The wzx and wzy genes in the O-antigen gene clusters of E. coli O22 and E. coli O91 were selected as target genes. The assays were serogroup-specific when tested against 72 E. coli O22 strains and 57 E. coli O91 strains isolated from food, humans, and animals, representative strains belonging to 168 E. coli O serogroups and non-E. coli bacteria. Furthermore, 72 E. coli O22 strains and 57 E. coli O91 strains isolated from food, water, animals, and humans were tested by the PCR for the presence of six and 19 virulence genes, respectively, associated with pathogenic E. coli strains. Based on the PCR screening results, multiplex PCR assays targeting the O22 wzy gene and the cnf-1 and sfa genes in E. coli O22 and the O91 wzy gene, conserved sequences of stx ₁ and stx ₂ genes, and the astA and cdt-III genes in E. coli O91 were developed to detect and identify pathogenic strains belonging to serogroups O22 and O91. Furthermore, E. coli O22 and O91 were detected by multiplex PCR assays targeting the wzx or wzy genes and conserved sequences of the stx ₁ and stx ₂ genes in ground beef samples inoculated with approximately two colony-forming units (CFU)/25 g after 18-h enrichment. The results demonstrate that the E. coli O22 and O91 wzx and wzy gene sequences were specific for the respective serogroups and can be used as diagnostic markers for rapid identification of these serogroups as an alternative to serotyping. The multiplex PCR assays targeting the O22 and O91 wzx and wzy genes and virulence genes can be used to identify and to detect pathogenic strains of these serogroups in food and fecal samples. Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

A reversible approach to two’s complement addition using a novel reversible TCG gate and its 4 dot 2 electron QCA architecture

Microsystem Technologies - Classical computers are already facing threshold limitations with CMOS getting restricted to clocking speeds in GHz range and alarming heat dissipation issues. Both these...     

Multiplex PCR detection of Shiga toxin-producing Escherichia coli strains belonging to serogroups O157, O103, O91, O113, O145, O111, and O26 experimentally inoculated in beef carcass swabs, beef trim, and ground beef

Numerous foodborne outbreaks are attributed to Shiga toxin-producing Escherichia coli (STEC) and have been recognized for causing gastrointestinal disease in humans. Beef products have been considered the principal source of STEC. A multiplex PCR assay enabling simultaneous detection of STEC O103, O91, O113, O145, O111, O157, and O26 was developed and evaluated in artificially contaminated beef carcass swabs, beef trim, and ground beef after overnight enrichment. Individual serogroups were experimentally inoculated at low (1 to 10 CFU/ml) and high (11 to 100 CFU/ml) levels, and with a cocktail of strains belonging to two, four, and six serogroups. There was no significant difference in detecting single STEC strains under the different conditions. Only when strains were combined were there significant differences in detection of all cocktail isolates in some of the beef products. To address this issue, four serogroups were experimentally inoculated together at three different estimated levels (10, 10(2), and 10(3) CFU/ml) in all three beef products. Results yielded no significant difference in detecting STEC at the three inoculation levels (10, 10(2), and 10(3) CFU/ml) in trim and carcass swabs, but there was a significant difference in detecting STEC at the lowest levels (10 and 10(2) CFU/ml) in the 80:20 nonirradiated ground beef, and in the detection of STEC in irradiated ground beef. The findings from this study could provide industry and government agencies with a tool to evaluate the prevalence and incidence of STEC in beef products and their processing environments.     

Crystallization of double crystalline symmetric diblock copolymers

We report dynamic Monte Carlo simulation results on the crystallization of double crystalline symmetric A-B diblock copolymer, wherein the melting temperature of A-block is higher than B-block. Crystallization of A-block precedes the crystallization of B-block upon cooling from a homogeneous melt. The morphological development is controlled by the interplay between crystallization and microphase separation. With increasing segregation strength, we observe a gradual decrease in crystallinity accompanying with smaller and thinner crystals. During crystallization, A-block crystallizes first and creates confinement for the crystallization of B-block. Thus, crystallization of B-block slows down influencing the overall crystal morphology. At higher segregation strength, due to the repulsive interaction between blocks, block junction is stretched out, which is reflected in the increased value of mean square radius of gyration. As a result, a large number of smaller size crystals form with less crystallinity. The onset of microphase separation shifts towards higher temperature with increasing segregation strength. Isothermal crystallization reveals that the transition pathways strongly depend on segregation strength. The value of Avrami index shows the formation of two dimensional lamellar crystals of both the blocks. Two-step (sequential), compared to one-step (coincident) isothermal crystallization, produces higher crystallinity in A-block, however, the crystallinity of B-block is almost identical in both the cases.     

Novel materials from unsaturated polyester resin/styrene/tung oil blends with high impact strengths and enhanced mechanical properties

A novel material was prepared through the blending of an unsaturated polyester resin/styrene mix with tung oil, which offered improved impact strength, creep resistance, modulus, and hardness. A nanoindentation technique was used to investigate the mechanical properties. With the incorporation of 1 wt % tung oil into the unsaturated polyester matrix, the impact strength, modulus, and hardness increased by 15, 20, and 41%, respectively. The impact‐fractured surfaces were examined with scanning electron microscopy. The dynamic mechanical analysis was performed with a nanoindentation technique. The storage and loss modulus values were determined under cyclic loading as a function of indentation. The flexural properties also significantly increased with the incorporation of tung oil. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Spectroscopic Differentiation and Quantification of Microorganisms in Apple Juice

ABSTRACT: A fast and easy-to-operate Fourier Transform Infrared (FTIR) spectrometry-based approach was developed for microbial differentiation and quantification in apple juice. Eight different microorganisms were evaluated: Enterobacter cloacae, Salmonella typhimurium, Enterobacteraerogenes, Salmonella choleraesuis, Serratia marcescens, Pseudomonas vulgaris, Vibrio cholerae , and Hafnia alvei . FTIR spectroscopy combined with chemometrics could differentiate the microorganisms studied at low concentration level of 10³ colony-forming units (CFU) /mL in apple juice. The chemometric models developed to count microorganisms in apple juice were validated by an independent test set consisting of 18 samples and correlated against plate counts satisfactorily up to a detection limit of 10³ CFU/mL.     

Spectroscopic quantification of bacteria using artificial neural networks

Fourier transform-infrared spectroscopy, in conjunction with artificial neural networks, has been used for identification and classification of selected foodborne pathogens. Five bacterial species (Enterococcus faecium, Salmonella Enteritidis, Bacillus cereus, Yersinia enterocolitica, Shigella boydii) and five Escherichia coli strains (O103, O55, O121, O30, O26) suspended in phosphate-buffered saline were enumerated to provide seven different concentrations ranging from 10(9) to 10(3) CFU/ ml. The trained artificial neural networks were then validated with an independent subset of samples and compared with the traditional plate count method. It was found that the concentration-based classification of the species was 100% correct and the strain-based classification was 90 to 100% accurate.     

Detection of Escherichia coli O157:H7 in Food Using Real-Time Multiplex PCR Assays Targeting the stx 1, stx 2, wzy O157, and the fliC h7 or eae Genes

Escherichia coli O157:H7 is an important foodborne pathogen, and foods of bovine origin and fresh produce have been linked to outbreaks. Real-time multiplex PCR assays were developed to detect E. coli O157:H7 in different foods. Apple cider and raw milk (25 ml) and ground beef and lettuce (25 g) were inoculated with 2 or 20 colony-forming units (CFU) of E. coli O157:H7 380-94 and subjected to enrichment in RapidChek E. coli O157:H7 broth at 42°C. One milliliter of the enrichments was removed at 8 and 20 h, and following DNA extraction, real-time multiplex PCR assays targeting the stx ₁, stx ₂, and wzy _O157 genes in combination with probes and primers targeting either the fliC _h7 or the eae genes were performed using OmniMix HS beads and the SmartCycler. The sensitivity of the real-time multiplex PCR assay was about 225 CFU/PCR. E. coli O157:H7 was detected (fluorescent signal generated for all gene targets) in apple cider, raw milk, lettuce and ground beef samples inoculated with 2 or 20 CFU/g or 25 ml after both 8 and 20 h of enrichment. Enrichments of uninoculated food samples were negative using the multiplex PCR targeting the stx ₁, stx ₂, wzy _O157, and eae genes; however, using the assay targeting the stx ₁, stx ₂, wzy _O157, and fliC _h7 gene combination, a positive result was always obtained for the fliC _h7 gene using uninoculated ground beef enrichments. Use of other primer sets targeting the fliC _h7 gene gave similar results. The real-time multiplex PCR assays targeting the stx ₁, stx ₂, eae, and wzy _O157 or the fliC _h7 genes are sensitive and specific and can be used for the detection of E. coli O157:H7 in food, except that the fliC _h7 gene may not be a suitable target for the detection of E. coli O157:H7 in ground beef.