scholarly journals An Engineered Reporter Phage for the Fluorometric Detection of Escherichia coli in Ground Beef

2021 ◽  
Vol 9 (2) ◽  
pp. 436
Author(s):  
Anqi Chen ◽  
Danhui Wang ◽  
Sam R. Nugen ◽  
Juhong Chen
Keyword(s):  
Ground Beef ◽  
Food Samples ◽  
Target Cells ◽  
Fluorometric Detection ◽  
Fluorogenic Substrate ◽  
Diagnostic Assays ◽  
E Coli ◽  
Biological Contamination ◽  
T7 Phage ◽  
Beta Galactosidase

Despite enhanced sanitation implementations, foodborne bacterial pathogens still remain a major threat to public health and generate high costs for the food industry. Reporter bacteriophage (phage) systems have been regarded as a powerful technology for diagnostic assays for their extraordinary specificity to target cells and cost-effectiveness. Our study introduced an enzyme-based fluorescent assay for detecting the presence of E. coli using the T7 phage engineered with the lacZ operon which encodes beta-galactosidase (β-gal). Both endogenous and overexpressed β-gal expression was monitored using a fluorescent-based method with 4-methylumbelliferyl β-d-galactopyranoside (MUG) as the substrate. The infection of E. coli with engineered phages resulted in a detection limit of 10 CFU/mL in ground beef juice after 7 h of incubation. In this study, we demonstrated that the overexpression of β-gal coupled with a fluorogenic substrate can provide a straightforward and sensitive approach to detect the potential biological contamination in food samples. The results also suggested that this system can be applied to detect E. coli strains isolated from environmental samples, indicating a broader range of bacterial detection.

scholarly journals Bacteriophages Reduce Experimental Contamination of Hard Surfaces, Tomato, Spinach, Broccoli, and Ground Beef by Escherichia coli O157:H7

2008 ◽  
Vol 74 (20) ◽  
pp. 6230-6238 ◽  
Author(s):  
Tamar Abuladze ◽  
Manrong Li ◽  
Marc Y. Menetrez ◽  
Timothy Dean ◽  
Andre Senecal ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Food Samples ◽  
Significant Treatment ◽  
E Coli ◽  
Virulent Strains ◽  
Buffer Control ◽  
And Control ◽  
Phosphate Buffered Saline ◽  
Control Samples

ABSTRACT A bacteriophage cocktail (designated ECP-100) containing three Myoviridae phages lytic for Escherichia coli O157:H7 was examined for its ability to reduce experimental contamination of hard surfaces (glass coverslips and gypsum boards), tomato, spinach, broccoli, and ground beef by three virulent strains of the bacterium. The hard surfaces and foods contaminated by a mixture of three E. coli O157:H7 strains were treated with ECP-100 (test samples) or sterile phosphate-buffered saline buffer (control samples), and the efficacy of phage treatment was evaluated by comparing the number of viable E. coli organisms recovered from the test and control samples. Treatments (5 min) with the ECP-100 preparation containing three different concentrations of phages (1010, 109, and 108 PFU/ml) resulted in statistically significant reductions (P = <0.05) of 99.99%, 98%, and 94%, respectively, in the number of E. coli O157:H7 organisms recovered from the glass coverslips. Similar treatments resulted in reductions of 100%, 95%, and 85%, respectively, in the number of E. coli O157:H7 organisms recovered from the gypsum board surfaces; the reductions caused by the two most concentrated phage preparations were statistically significant. Treatment with the least concentrated preparation that elicited significantly less contamination of the hard surfaces (i.e., 109 PFU/ml) also significantly reduced the number of viable E. coli O157:H7 organisms on the four food samples. The observed reductions ranged from 94% (at 120 ± 4 h posttreatment of tomato samples) to 100% (at 24 ± 4 h posttreatment of spinach samples). The data suggest that naturally occurring bacteriophages may be useful for reducing contamination of various hard surfaces, fruits, vegetables, and ground beef by E. coli O157:H7.

Simultaneous Immunomagnetic Separation Method for the Detection of Escherichia coli O26, O111, and O157 from Food Samples

2014 ◽  
Vol 77 (1) ◽  
pp. 15-22 ◽  
Author(s):  
MASASHI KANKI ◽  
KAZUKO SETO ◽  
YUKO KUMEDA
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Food Samples ◽  
Food Culture ◽  
Cell Detection ◽  
Labor Costs ◽  
Single Strain ◽  
E Coli ◽  
Detection Medium

We performed a simultaneous immunomagnetic separation (IMS) assay on Shiga toxin–producing Escherichia coli (STEC) serogroups O26, O111, and O157 with immunobeads coated with O26, O111, or O157 antibodies that were simultaneously added to an aliquot of food culture. We also compared the usefulness of CHROMagar STEC medium against various selective isolation agars designed to test for the three serogroups. Samples of sliced beef, ground beef, and radish sprout were artificially contaminated with STEC O26, O111, and O157 strains after incubation in enrichment broth and were subjected to conventional and simultaneous IMS assays. Simultaneous IMS did not affect the sensitivity of target cell detection. For STEC O26, O111, and O157 inoculated into the enriched samples of sliced beef and radish sprout, the detection ability of CHROMagar STEC was similar to or exceeded that of other isolation agars. However for STEC O157 inoculated into ground beef cultures, cefixime tellurite sorbitol MacConkey agar (CT-SMAC) was the superior detection medium. The performance of simultaneous IMS combined with CT-SMAC and CHROMagar STEC detection is similar to that of the Japanese standard method for isolating E. coli O26, O111, and O157. However, the proposed approach involves the same time, materials, and labor costs as the standard E. coli O157 reference procedure but allows detection of three E. coli serotypes rather than a single strain.

Incidence of Enterohemorrhagic Escherichia coli, Escherichia coli O157, Salmonella, and Listeria monocytogenes in Retail Fresh Ground Beef, Sprouts, and Mushrooms

2006 ◽  
Vol 69 (2) ◽  
pp. 441-443 ◽  
Author(s):  
M. SAMADPOUR ◽  
M. W. BARBOUR ◽  
T. NGUYEN ◽  
T.-M. CAO ◽  
F. BUCK ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Ground Beef ◽  
Food Samples ◽  
Enterohemorrhagic Escherichia Coli ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Pcr Assays ◽  
Retail Food

The objective of this study was to determine the prevalence of enterohemorrhagic Escherichia coli (EHEC), E. coli O157, Salmonella, and Listeria monocytogenes in retail food samples from Seattle, Wash. A total of 2,050 samples of ground beef (1,750 samples), mushrooms (100 samples), and sprouts (200 samples) were collected over a 12-month period and analyzed for the presence of these pathogens. PCR assays, followed by culture confirmation were used to determine the presence or absence of each organism. Of the 1,750 ground beef samples analyzed, 61 (3.5%) were positive for EHEC, and 20 (1.1%) of these were positive for E. coli O157. Salmonella was present in 67 (3.8%) of the 1,750 ground beef samples. Of 512 ground beef samples analyzed, 18 (3.5%) were positive for L. monocytogenes. EHEC was found in 12 (6.0%) of the 200 sprout samples, and 3 (1.5%) of these yielded E. coli O157. Of the 200 total sprout samples, 14 (7.0%) were positive for Salmonella and none were positive for L. monocytogenes. Among the 100 mushroom samples, 4 (4.0%) were positive for EHEC but none of these 4 samples were positive for E. coli O157. Salmonella was detected in 5 (5.0%) of the mushroom samples, and L. monocytogenes was found in 1 (1.0%) of the samples.

Enumeration of Sublethally Injured Escherichia coli O157:H7 ATCC 43895 and Escherichia coli Strain B-41560 Using Selective Agar Overlays versus Commercial Methods

2013 ◽  
Vol 76 (4) ◽  
pp. 674-679 ◽  
Author(s):  
AMANDA R. SMITH ◽  
ALYSHA L. ELLISON ◽  
AMANDA L. ROBINSON ◽  
MARYANNE DRAKE ◽  
SUSAN A. McDOWELL ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Food Samples ◽  
Coli Strain ◽  
Milk Formula ◽  
Selective Media ◽  
E Coli ◽  
Infant Milk Formula ◽  
Intervention Measures ◽  
Direct Inoculation

Quality control procedures during food processing may involve direct inoculation of food samples onto appropriate selective media for subsequent enumeration. However, sublethally injured bacteria often fail to grow, enabling them to evade detection and intervention measures and ultimately threaten the health of consumers. This study compares traditional selective and nonselective agar-based overlays versus two commercial systems (Petrifilm and Easygel) for recovery of injured E. coli B-41560 and O157:H7 strains. Bacteria were propagated in tryptic soy broth (TSB), ground beef slurry, and infant milk formula to a density of 106 to 108 CFU/ml and then were stressed for 6 min either in lactic acid (pH 4.5) or heat shocked for 3 min at 60°C. Samples were pour plated in basal layers of either tryptic soy agar (TSA), sorbitol MacConkey agar (SMAC), or violet red bile agar (VRB) and were resuscitated for 4 h prior to addition of agar overlays. Other stressed bacteria were plated directly onto Petrifilm and Easygel. Results indicate that selective and nonselective agar overlays recovered significantly higher numbers (greater than 1 log) of acid-and heat-injured E. coli O157:H7 from TSB, ground beef, and infant milk formula compared with direct plating onto selective media, Petrifilm, or Easygel, while no significant differences among these media combinations were observed for stressed E. coli B-41560. Nonstressed bacteria from TSB and ground beef were also recovered at densities significantly higher in nonselective TSA-TSA and in VRB-VRB and SMAC-SMAC compared with Petrifilm and Easygel. These data underscore the need to implement food safety measures that address sublethally injured pathogens such as E. coli O157:H7 in order to avoid underestimation of true densities for target pathogens.

scholarly journals USDA FSIS, FDA BAM, and ISO Culture Methods BD BBL CHROMagar O157 Media

2009 ◽  
Vol 92 (4) ◽  
pp. 1118-1127 ◽  
Author(s):  
Vicki Ritter ◽  
Susan Kircher ◽  
Nancy Dick
Keyword(s):  
Ground Beef ◽  
Reference Method ◽  
Food Samples ◽  
Culture Methods ◽  
Chi Square ◽  
Apple Cider ◽  
E Coli ◽  
Inspection Service ◽  
Chi Square Analysis ◽  
Method Agreement

Abstract BBL CHROMagar O157 media (CO) was evaluated for detection of Escherichia coli O157:H7 in raw ground beef and unpasteurized apple cider. The recovery of E. coli O157:H7 on CO was compared to the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM), U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS), and International Organization for Standardization (ISO) reference-plated media using the recommended enrichment broths. Of the 180 food samples tested, 45 were tested using BAM, 45 using the USDA method, and 90 using the ISO method. CO produced comparable results with the reference methods on all matrixes with a sensitivity of 100 and a specificity of 100. No false negatives were found in testing the food matrixes. There was no statistical difference in recovery based on Chi-square analysis. Method agreement for raw ground beef was 85 for the USDA FSIS method and 95 for the ISO method. Method agreement for unpasteurized apple cider was 100 for the ISO and FDA BAM methods. In all cases where method agreement was &lt;100, CO detected more positives than the reference method media. Evaluation of known isolates on CO in inclusivity and exclusivity testing had a sensitivity and specificity of 100. The results of this study demonstrate that CO is an effective medium for the recovery and detection of E. coli O157:H7 in raw ground beef and unpasteurized apple cider using FDA BAM, USDA FSIS, and ISO methods.

A New Fluorogenic Substrate Method for Assay of Bacterial Endotoxins Using Limulus Hemocyte Lysate

1979 ◽  
Author(s):  
T Harada ◽  
M Ohki ◽  
M Niwa ◽  
S Iwanaga
Keyword(s):  
Antithrombin Iii ◽  
Assay Method ◽  
Sensitive Assay ◽  
Fluorogenic Substrate ◽  
Toxin Concentration ◽  
Amidase Activity ◽  
E Coli ◽  
Bacterial Endotoxins ◽  
Plasmin Inhibitor ◽  
Limulus Test

Limulus hemocyte lysate contains a proclotting enzyme, which is transformed to the active clotting enzyme in the presence of gram-negative bacterial endotoxins. The clotting enzyme coagulates a clottable protein, named coagulogen, contained also in the lysate. This gelation reaction of the lysate, named Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. We developed a new fluorogenic substrate, Boc-Leu-Gly-Arg-4-methylcoumarin amide, for Limulus clotting enzyme and established an enzymatic assay method for endotoxins, using the substrate. Because the endotoxin mediates the activation of proclotting enzyme in the lysate, the measurement of amidase activity could be applicable for quantitation of the endotoxins. In fact, the amidase activity determined fluorometrically increased by increasing concentration of E. coli 0111: B4 endotoxin added to the lysate, and a linear relationship between the toxin concentration and the activity was observed in the range of 5X10-6to 5xl0-2 µg endotoxin. The method was a fifty times more sensitive than that of the Limulus test and was very reproducible. However, the method was not directly applicable for the assay of endotoxins in circulating blood, as the amidase activity was strongly inhibited by antithrombin III and α2-plasmin inhibitor. Thus, some pretreatment with heat or chloroform on plasma samples before the assay was required.

Cell-Specific Chemical Delivery Using a Selective Nitroreductase–Nitroaryl Pair

2018 ◽  
Author(s):  
Todd D. Gruber ◽  
Chithra Krishnamurthy ◽  
Jonathan B. Grimm ◽  
Michael R. Tadross ◽  
Laura M. Wysocki ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Target Cells ◽  
Specific Chemical ◽  
E Coli ◽  
Enzyme Substrate ◽  
Cell Specificity ◽  
General Chemical ◽  
Increased Activity ◽  
Enzyme Variant

<p>The utility of<b> </b>small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. ‘Masking’ the activity of small molecules using a general chemical modification and ‘unmasking’ it only within target cells could overcome this limitation. To this end, we have developed a selective enzyme–substrate pair consisting of engineered variants of <i>E. coli</i> nitroreductase (NTR) and a 2‑nitro-<i>N</i>-methylimidazolyl (NM) masking group. To discover and optimize this NTR–NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.</p>

scholarly journals Comparison of Fecal Coliform Agar and Violet Red Bile Lactose Agar for Fecal Coliform Enumeration in Foods

2002 ◽  
Vol 68 (4) ◽  
pp. 1631-1638 ◽  
Author(s):  
A. Leclercq ◽  
C. Wanegue ◽  
P. Baylac
Keyword(s):  
Escherichia Coli ◽  
Fecal Coliform ◽  
Food Samples ◽  
Fecal Coliforms ◽  
Predictive Values ◽  
Direct Plating ◽  
Hafnia Alvei ◽  
E Coli ◽  
Plating Method ◽  
Mashed Potatoes

ABSTRACT A 24-h direct plating method for fecal coliform enumeration with a resuscitation step (preincubation for 2 h at 37 ± 1°C and transfer to 44 ± 1°C for 22 h) using fecal coliform agar (FCA) was compared with the 24-h standardized violet red bile lactose agar (VRBL) method. FCA and VRBL have equivalent specificities and sensitivities, except for lactose-positive non-fecal coliforms such as Hafnia alvei, which could form typical colonies on FCA and VRBL. Recovery of cold-stressed Escherichia coli in mashed potatoes on FCA was about 1 log unit lower than that with VRBL. When the FCA method was compared with standard VRBL for enumeration of fecal coliforms, based on counting carried out on 170 different food samples, results were not significantly different (P > 0.05). Based on 203 typical identified colonies selected as found on VRBL and FCA, the latter medium appears to allow the enumeration of more true fecal coliforms and has higher performance in certain ways (specificity, sensitivity, and negative and positive predictive values) than VRBL. Most colonies clearly identified on both media were E. coli and H. alvei, a non-fecal coliform. Therefore, the replacement of fecal coliform enumeration by E. coli enumeration to estimate food sanitary quality should be recommended.

scholarly journals Lessons from a large outbreak of Escherichia coli O157[ratio ]H7 infections: insights into the infectious dose and method of widespread contamination of hamburger patties

1999 ◽  
Vol 122 (2) ◽  
pp. 185-192 ◽  
Author(s):  
J. TUTTLE ◽  
T. GOMEZ ◽  
M. P. DOYLE ◽  
J. G. WELLS ◽  
T. ZHAO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Most Probable Number ◽  
Strong Argument ◽  
Probable Number ◽  
Infectious Dose ◽  
E Coli ◽  
Beef Patties ◽  
Large Outbreak ◽  
Microbiological Testing

Between November 1992 and February 1993, a large outbreak of Escherichia coli O157[ratio ]H7 infections occurred in the western USA and was associated with eating ground beef patties at restaurants of one fast-food chain. Restaurants that were epidemiologically linked with cases served patties produced on two consecutive dates; cultures of recalled ground beef patties produced on those dates yielded E. coli O157[ratio ]H7 strains indistinguishable from those isolated from patients, confirming the vehicle of illness. Seventy-six ground beef patty samples were cultured quantitatively for E. coli O157[ratio ]H7. The median most probable number of organisms was 1·5 per gram (range, <0·3–15) or 67·5 organisms per patty (range, <13·5–675). Correlation of the presence of E. coli O157[ratio ]H7 with other bacterial indicators yielded a significant association between coliform count and the presence of E. coli O157[ratio ]H7 (P=0·04). A meat traceback to investigate possible sources of contamination revealed cattle were probably initially colonized with E. coli O157[ratio ]H7, and that their slaughter caused surface contamination of meat, which once combined with meat from other sources, resulted in a large number of contaminated ground beef patties. Microbiological testing of meat from lots consumed by persons who became ill was suggestive of an infectious dose for E. coli O157[ratio ]H7 of fewer than 700 organisms. These findings present a strong argument for enforcing zero tolerance for this organism in processed food and for markedly decreasing contamination of raw ground beef. Process controls that incorporate microbiological testing of meat may assist these efforts.

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