Home About us Contact
|
Home About us Contact | ||
|
|
||
H7 Cells (h7 + cell)
Selected AbstractsDetection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCRJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2009L. Wang Abstract Aims:, The aim of this study was to develop and optimize a novel method that combines ethidium bromide monoazide (EMA) staining with real-time PCR for the detection of viable Escherichia coli O157:H7 in ground beef. EMA can penetrate dead cells and bind to intracellular DNA, preventing its amplification via PCR. Methods and Results:, Samples were stained with EMA for 5 min, iced for 1 min and exposed to bright visible light for 10 min prior to DNA extraction, to allow EMA binding of the DNA from dead cells. DNA was then extracted and amplified by TaqManŽ real-time PCR to detect only viable E. coli O157:H7 cells. The primers and TaqManŽ probe used in this study target the uidA gene in E. coli O157:H7. An internal amplification control (IAC), consisting of 0ˇ25 pg of plasmid pUC19, was added in each reaction to prevent the occurrence of false-negative results. Results showed a reproducible application of this technique to detect viable cells in both broth culture and ground beef. EMA, at a final concentration of 10 ,g ml,1, was demonstrated to effectively bind DNA from 108 CFU ml,1 dead cells, and the optimized method could detect as low as 104 CFU g,1 of viable E. coli O157:H7 cells in ground beef without interference from 108 CFU g,1 of dead cells. Conclusions:, EMA real-time PCR with IAC can effectively separate dead cells from viable E. coli O157:H7 and prevent amplification of DNA in the dead cells. Significance and Impact of the Study:, The EMA real-time PCR has the potential to be a highly sensitive quantitative detection technique to assess the contamination of viable E. coli O157:H7 in ground beef and other meat or food products. [source] Comparison of ATP and in vivo bioluminescence for assessing the efficiency of immunomagnetic sorbents for live Escherichia coli O157:H7 cellsJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2002W. Sun Aims:,To develop methods to assess the efficiency of immunomagnetic separation (IMS). Methods and Results:,The capturing efficiency of biosorbents for Escherichia coli O157:H7, constructed using streptavidin-coated magnetic beads and biotinylated antibodies, was tested using both in vivo and ATP bioluminescence. Both methods were suitable for the enumeration of bacteria captured by the biosorbents. The level of both ATP and in vivo bioluminescence depended on the media used, but was unaffected by the magnetic beads. The capture efficiency depended on time and sample volume, but did not depend on the length of spacer arm of the biotinylation agent. For cell concentrations of , 105 cfu ml,1, in a 1-ml sample volume, nearly 80,85% recovery of the pathogen was observed after 0ˇ5 h of incubation. For an 11-ml sample containing 104 cfu ml,1, maximum recovery (50% of cells) was achieved only after 2 h incubation. Conclusions:,The detection limit of an ATP-based bioluminescent assay for E. coli O157:H7 was reduced by 1 log cycle after optimization of IMS. The bioluminescent methods could be used for screening and testing the affinity of antibodies or other affinity elements of biosorbents towards live bacterial cells. Significance and Impact of the Study:,Bioluminescent assays provide an easy way to optimize conditions for the capture of bacteria by biosorbents in real time. [source] Whole-Leaf Wash Improves Chlorine Efficacy for Microbial Reduction and Prevents Pathogen Cross-Contamination during Fresh-Cut Lettuce ProcessingJOURNAL OF FOOD SCIENCE, Issue 5 2010Xiangwu Nou Abstract:, Currently, most fresh-cut processing facilities in the United States use chlorinated water or other sanitizer solutions for microbial reduction after lettuce is cut. Freshly cut lettuce releases significant amounts of organic matter that negatively impacts the effectiveness of chlorine or other sanitizers for microbial reduction. The objective of this study is to evaluate whether a sanitizer wash before cutting improves microbial reduction efficacy compared to a traditional postcutting sanitizer wash. Romaine lettuce leaves were quantitatively inoculated with,E. coli,O157:H7 strains and washed in chlorinated water before or after cutting, and,E. coli,O157:H7 cells that survived the washing process were enumerated to determine the effectiveness of microbial reduction for the 2 cutting and washing sequences. Whole-leaf washing in chlorinated water improved pathogen reduction by approximately 1 log unit over traditional cut-leaf sanitization. Similar improvement in the reduction of background microflora was also observed. Inoculated "Lollo Rossa" red lettuce leaves were mixed with noninoculated Green-Leaf lettuce leaves to evaluate pathogen cross-contamination during processing. High level (96.7% subsamples, average MPN 0.6 log CFU/g) of cross-contamination of noninoculated green leaves by inoculated red leaves was observed when mixed lettuce leaves were cut prior to washing in chlorinated water. In contrast, cross-contamination of noninoculated green leaves was significantly reduced (3.3% of subsamples, average MPN ,,0.3 log CFU/g) when the mixed leaves were washed in chlorinated water before cutting. This result suggests that whole-leaf sanitizing washes could be a practical strategy for enhancing the efficacy of chlorine washes for pathogen reduction and cross-contamination prevention. Practical Application:, Freshly cut leafy greens release large amount of organic matter that negatively impact the chlorine washing efficacy. Implementing the primary antimicrobial intervention step of chlorine washing prior to cutting can significantly improve the efficacy of microbial reduction and minimize pathogen cross-contamination. [source] A Recombinant Bacteriophage-Based Assay for the Discriminative Detection of Culturable and Viable but Nonculturable Escherichia coli O157:H7BIOTECHNOLOGY PROGRESS, Issue 3 2006Raheela Awais A previously green fluorescent protein (GFP)-labeled PP01 virulent bacteriophage, specific to Escherichia coli O157:H7, was used to construct lysozyme-inactivated GFP-labeled PP01 phage (PP01e - /GFP). The new recombinant phage lacked lytic activity because of the inactivation of gene e, which produces the lysozyme responsible for cell lysis. Gene e was inactivated by inserting an amber stop codon. Prolonged incubation ofE. coli O157:H7 cells with PP01e - /GFP did not lead to cell lysis, while the propagation of PP01e - /GFP in host cells increased the intensity of green fluorescence. Retention of cell morphology and increase in fluorescence enabled the direct visualization and enumeration of E. coli O157:H7 cells within an hour. The PP01e - /GFP system, when combined with nutrient uptake analysis, further allowed the discriminative detection of culturable, viable but nonculturable (VBNC), and dead cells in the stress-induced aquatic environment. Stress-induced cells, which retained culturability, allowed phage propagation and produced bright green florescence. Nonculturable cells (VBNC and dead) allowed only phage adsorption but no proliferation and remained low fluorescent. The low-fluorescent nonculturable cells were further differentiated into VBNC and dead cells on the basis of nutrient uptake analysis. The low-fluorescent cells, which grew in size by nutrient incorporation during prolonged incubation in nutrient medium, were defined as metabolically active and in the VBNC state. The elongated VBNC cells were then easily recognizable from dead cells. The proposed assay enabled the detection and quantification of VBNC cells. Additionally, it revealed the proportion of culturable to VBNC cells within the population, as opposed to conventional techniques, which demonstrate VBNC cells as a differential value of the total viable count and the culturable cell count. [source] | ||||||||||