Specific Identification of Escherichia coli O157 by an Immunostick Method Using Commercially Available Antibodies

1996 ◽  
Vol 59 (6) ◽  
pp. 670-673 ◽  
Author(s):  
SHIU W. HUANG ◽  
TSUNG C. CHANG
Keyword(s):  
Escherichia Coli ◽  
Rapid Identification ◽  
Escherichia Coli O157 ◽  
Commercial Preparation ◽  
E Coli ◽  
Specificity And Sensitivity ◽  
Sandwich Enzyme Immunoassay ◽  
Antigen Antibody ◽  
Phosphate Buffered Saline ◽  
Somatic Antigens

A sandwich enzyme-immunoassay performed on plastic sticks was developed to specifically identify Escherichia coli O157. Colonies of test bacteria grown on tryptic soy agar were suspended in phosphate-buffered saline, heated in a 100°C water bath for 15 min, and incubated with the plastic sticks coated with a commercial preparation of anti-E. coli O157 antibodies at 37°C for 1.5 h. After incubation, the same antibodies labeled with peroxidase were used to produce the signal of antigen-antibody reaction. For 35 strains of E. coli O157 (among them 34 were E. coli O157:H7) tested, all produced strong reactions by the immunoassay. For 162 strains of E. coli with somatic antigens other than O157 and 38 strains of other genera tested, only one strain (Salmonella bietri) produced a false-positive reaction. The specificity and sensitivity of the immunostick assay were 100% (35/35) and 99.5% (199/200), respectively. The detection limit of the assay for E. coli O157:H7 (CCRC 15991) was about 105 CFU/ml. The method, which can be carried out within 3 h, is useful for rapid identification of suspect E. coli O157 isolated on selective media.

Factors Influencing Attachment of Escherichia coli O157:H7 to Beef Tissues and Removal Using Selected Sanitizing Rinses‡

1996 ◽  
Vol 59 (5) ◽  
pp. 453-459 ◽  
Author(s):  
PINA M. FRATAMICO ◽  
FRANKIE J. SCHULTZ ◽  
ROBERT C. BENEDICT ◽  
ROBERT L. BUCHANAN ◽  
PETER H. COOKE
Keyword(s):  
Escherichia Coli ◽  
Acetic Acid ◽  
Surface Structures ◽  
Escherichia Coli O157 ◽  
Adipose Tissues ◽  
E Coli ◽  
Factors Influencing ◽  
Significant Difference ◽  
Phosphate Buffered Saline ◽  
Scanning Electron

Attachment of E. coli O157:H7 and E. coli K12 to beef tenderloin filet, chuck, and adipose tissues was studied. Most attachment occurred within 1 min of incubation; the number of attached organisms depended on the concentration of bacteria in the liquid inoculum. Similar levels of E. coli bound to the three types of beef tissues tested. E. coli O157:H7 was heavily piliated; however, there was no significant difference between levels of bound E. coli O157:H7 and E. coli K12, indicating that these surface structures apparently are not involved in attachment. Scanning electron photomicrographs of meat tissue and of purified collagen suggested that bacteria attached primarily to collagen fibers. Rinsing solutions consisting of 10% trisodium phosphate (TSP), 2% acetic acid (HAc), phosphate-buffered saline (PBS) and combinations of each were tested for effectiveness in reducing the number of attached E. coli. The level of bacteria removed from tenderloin tissue following TSP, HAc, or PBS rinses did not differ considerably. When beef tissues were stored at 4°C for 18 h after the various rinse combinations, TSP rinse treatments reduced the levels of E. coli K12 and O157:H7 attached to adipose tissue up to 3.4 and 2.7 log units, respectively, compared to PBS rinse treatments. Therefore, TSP may be effective for reducing populations of E. coli O157:H7 on beef carcass tissue.

Heat Resistance of Escherichia coli O157:H7 in a Nutrient Medium and in Ground Beef Patties as Influenced by Storage and Holding Temperatures

1996 ◽  
Vol 59 (3) ◽  
pp. 230-237 ◽  
Author(s):  
TIMOTHY C. JACKSON ◽  
MARGARET D. HARDIN ◽  
GARY R. ACUFF
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Heat Resistance ◽  
Elevated Temperatures ◽  
Potential Temperature ◽  
Ground Beef ◽  
Escherichia Coli O157 ◽  
Commercial Preparation ◽  
E Coli ◽  
Beef Patties

Stationary-phase cultures of Escherichia coli O157:H7 were inoculated into tryptic soy broth, sealed in vials, and stored at −18°C for 1, 8, and 15 days, or 3 or 15°C for 3, 6, and 9 h. Thermal resistance was determined at 55°C. Each storage treatment was repeated with additional holding at 23 or 30°C for 1, 2, 3, or 4 h prior to heating to simulate potential temperature abuse during handling. Cultures under treatments enabling the growth of E. coli O157:H7 were generally more heat sensitive than those held at temperatures which restricted growth or enabled growth to stationary phase. Cultures stored frozen (−18°C) without holding at elevated temperatures had greater heat resistance than those stored under refrigeration (3°C) or at 15°C. Subsequent holding of frozen cultures at 23 or 30°C resulted in a decrease in heat resistance. To determine whether these responses would be observed under typical commercial preparation procedures, ground beef patties were inoculated with E. coli O157:H7 and stored at 3 or 15°C for 9 h or at −18°C for 8 d and then held at 21 or 30°C for 0 or 4 h. Patties were grilled to an internal temperature of 54.4°C (130°F), 62.8°C (145°F), or 68.3°C (155°F). Cultures were most resistant in frozen patties, while cultures in patties stored at 15°C were the most heat sensitive. Holding patties at 21 or 30°C prior to grilling resulted in increased sensitivity. Storage and holding temperatures similar to those encountered in food service may influence the ability of E. coli O157:H7 to survive heat treatments.

Development of a Rapid Immunochromatographic Strip for Detection of Escherichia coli O157

2005 ◽  
Vol 68 (10) ◽  
pp. 2140-2143 ◽  
Author(s):  
BYEONG YEAL JUNG ◽  
SUK CHAN JUNG ◽  
CHANG HEE KWEON
Keyword(s):  
Escherichia Coli ◽  
High Specificity ◽  
Escherichia Coli O157 ◽  
Test Results ◽  
Positive Signal ◽  
Immunochromatographic Strip ◽  
Selective Enrichment ◽  
E Coli ◽  
Specificity And Sensitivity ◽  
Strip Test

We developed an immunochromatographic (IC) strip for the rapid detection of Escherichia coli O157 in enriched samples. Murine monoclonal antibody to E. coli O157:H7 lipopolysaccharide was conjugated with 40 nm of colloidal gold particles by the citrate method. The specificity of the IC strip was determined using 48 pure-cultured bacteria, including 32 E. coli strains and 16 non–E. coli strains. Regardless of H serotype, E. coli O157 strains produced a positive signal, whereas the others, representing 29 E. coli serotypes, did not. Among 16 non–E. coli strains, only Citrobacter amalonaticus yielded a positive signal. The sensitivity of the IC strip was determined using 10-fold diluted E. coli O157:H7, with a range of 1.8 × 107 to 1.8 CFU/ml in enriched raw beef. E. coli O157 could be detected at a minimum of 1.8 × 105 CFU/ml without enrichment and 1.8 CFU/ml after enrichment. Various samples were enriched to detect E. coli O157 using the IC strip and to isolate E. coli O157:H7 using traditional culture procedures. The IC strip test results exhibited 100% agreement with traditional methods after selective enrichment, since E. coli O157:H7 was also isolated from all the samples with positive strip test results. However, the specificity of the strip was somewhat higher with pork (98.8%) than with bovine feces (87.9%) and swine feces (93.4%). These results indicated that the IC strip exhibits high specificity and sensitivity in the detection of E. coli O157, and this assay is rapid, economical, and simple, without requirement of complicated equipment.

scholarly journals Human infections with verocytotoxin-producing Escherichia coli O157 – 10 years of E. coli O157 serodiagnosis

2008 ◽  
Vol 57 (11) ◽  
pp. 1389-1393 ◽  
Author(s):  
Henrik Chart ◽  
Thomas Cheasty
Keyword(s):  
Escherichia Coli ◽  
Haemolytic Uraemic Syndrome ◽  
Enteric Pathogens ◽  
Escherichia Coli O157 ◽  
Health Protection Agency ◽  
Protection Agency ◽  
E Coli ◽  
Males And Females ◽  
Human Infections ◽  
Somatic Antigens

From 1997 to 2007, the Laboratory of Enteric Pathogens (LEP), Health Protection Agency, UK, received sera from 2148 patients for testing for antibodies to the LPS of verocytotoxin-producing Escherichia coli (VTEC) O157. A total of 676 (31.5 %) sera had antibodies binding the LPS of E. coli O157 and the majority of patients were below the age of 10 years, a trend observed for both males and females. Antibody-positive patients had haemolytic uraemic syndrome (HUS) in 79.3 % of cases and most of these presented with the atypical (D−) form of HUS. Nine patients were shown to have antibodies to the LPS of E. coli belonging to serogroups O26 (4), O103 (2), O111 (1) and O145 (2) and one patient had antibodies to the somatic antigens of both E. coli O26 and O103. The serodiagnosis of infections with E. coli O157 and other VTEC continues to be an important adjunct to bacteriology. Where clinicians suspect the involvement of a VTEC in disease, patients' sera should be submitted to the LEP for analysis without delay.

Evaluation of Culture- and PCR-Based Detection Methods for Escherichia coli O157:H7 in Inoculated Ground Beef†

2005 ◽  
Vol 68 (8) ◽  
pp. 1566-1574 ◽  
Author(s):  
TERRANCE M. ARTHUR ◽  
JOSEPH M. BOSILEVAC ◽  
XIANGWU NOU ◽  
MOHAMMAD KOOHMARAIE
Keyword(s):  
Escherichia Coli ◽  
False Positive ◽  
False Negative ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Perishable Product ◽  
Specificity And Sensitivity

Currently, several beef processors employ test-and-hold systems for increased quality control of ground beef. In such programs, each lot of product must be tested and found negative for Escherichia coli O157:H7 prior to release of the product into commerce. Optimization of three testing attributes (detection time, specificity, and sensitivity) is critical to the success of such strategies. Because ground beef is a highly perishable product, the testing methodology used must be as rapid as possible. The test also must have a low false-positive result rate so product is not needlessly discarded. False-negative results cannot be tolerated because they would allow contaminated product to be released and potentially cause disease. In this study, two culture-based and three PCR-based methods for detecting E. coli O157:H7 in ground beef were compared for their abilities to meet the above criteria. Ground beef samples were individually spiked with five genetically distinct strains of E. coli O157: H7 at concentrations of 17 and 1.7 CFU/65 g and then subjected to the various testing methodologies. There was no difference (P > 0.05) in the abilities of the PCR-based methods to detect E. coli O157:H7 inoculated in ground beef at 1.7 CFU/65 g. The culture-based systems detected more positive samples than did the PCR-based systems, but the detection times (21 to 48 h) were at least 9 h longer than those for the PCR-based methods (7.5 to 12 h). Ground beef samples were also spiked with potentially cross-reactive strains. The PCR-based systems that employed an immunomagnetic separation step prior to detection produced fewer false-positive results.

Attachment and Biofilm Formation by Escherichia coli O157:H7 on Stainless Steel as Influenced by Exopolysaccharide Production, Nutrient Availability, and Temperature

2004 ◽  
Vol 67 (10) ◽  
pp. 2123-2131 ◽  
Author(s):  
JEE-HOON RYU ◽  
HOIKYUNG KIM ◽  
LARRY R. BEUCHAT
Keyword(s):  
Escherichia Coli ◽  
Stainless Steel ◽  
Nutrient Availability ◽  
Biofilm Formation ◽  
Escherichia Coli O157 ◽  
Strain Atcc ◽  
Wild Type ◽  
E Coli ◽  
Nutrient Environment ◽  
Phosphate Buffered Saline

The influence of exopolysaccharide (EPS) production, nutrient availability, and temperature on attachment and biofilm formation by Escherichia coli O157:H7 strains ATCC 43895 (wild type) and 43895-EPS (extensive EPS-producing mutant) on stainless steel coupons (SSCs) was investigated. Cells grown on heated lettuce juice agar and modified tryptic soy agar were suspended in phosphate-buffered saline (PBS). SSCs were immersed in the cell suspension (109 CFU/ml) at 4°C for 24 h. Biofilm formation by cells attached to SSCs as affected by immersing in 10% tryptic soy broth (TSB), lettuce juice broth (LJB), and minimal salts broth (MSB) at 12 and 22°C was studied. A significantly lower number of strain 43895-EPS cells, compared to strain ATCC 43895 cells, attached to SSCs during a 24-h incubation (4°C) period in PBS suspension. Neither strain formed a biofilm on SSCs subsequently immersed in 10% TSB or LJB, but both strains formed biofilms in MSB. Populations of attached cells and planktonic cells of strain ATCC 43895 gradually decreased during incubation for 6 days in LJB at 22°C, but populations of strain 43895-EPS remained constant for 6 days at 22°C, indicating that the EPS-producing mutant, compared to the wild-type strain, has a higher tolerance to the low-nutrient environment presented by LJB. It is concluded that EPS production by E. coli O157:H7 inhibits attachment to SSCs and that reduced nutrient availability enhances biofilm formation. Biofilms formed under conditions favorable for EPS production may protect E. coli O157:H7 against sanitizers used to decontaminate lettuce and produce processing environments. Studies are under way to test this hypothesis.

Reduction of Escherichia coli O157:H7 Populations in Soy Sauce, a Fermented Seasoning

1998 ◽  
Vol 61 (6) ◽  
pp. 657-661 ◽  
Author(s):  
SUSUMU MASUDA ◽  
YUKIKO HARA-KUDO ◽  
SUSUMU KUMAGAI
Keyword(s):  
Escherichia Coli ◽  
Butyl Ester ◽  
Soy Sauce ◽  
Hydroxybenzoic Acid ◽  
Escherichia Coli O157 ◽  
Undetectable Level ◽  
Control Solution ◽  
E Coli ◽  
Cell Numbers ◽  
Phosphate Buffered Saline

We studied five Escherichia coli O157:H7 strains in soy sauce which was incubated at 4, 18, or 30°C after inoculation. The cell numbers of E. coli O157:H7 decreased to an undetectable level (<20 CFU/ml) within 9 days in all the soy sauce samples at 30°C, but did not decrease in the 0.1 M phosphate-buffered saline (pH 7.0) control solution under the same conditions. Soy sauce reduced the cell numbers of bacteria at 18°C to a lesser extent than at 30°C, but to a greater extent than at 4°C. Components of soy sauce such as 10% or 16% NaCl, 5% ethanol, lactic acid, or acetic acid at pH 4.5, sodium benzoate (0.6 g/kg), or p-hydroxybenzoic acid n-butyl ester (0.05 g/liter) caused a reduction of the E. coli O157:H7 population at 30°C, and the anti-E. coli O157:H7 effect of each component was less than that of soy sauce. The fate of E. coli O157:H7 cells in a buffered solution containing various components of soy sauce resembled that in soy sauce at 30°C, which demonstrated the importance of the combination of the soy sauce components for its anti-E. coli O157:H7 action.

Occurrence of Shiga Toxin–Producing Escherichia coli O157 in Selected Dairy and Meat Products Marketed in the City of Rabat, Morocco

2004 ◽  
Vol 67 (6) ◽  
pp. 1234-1237 ◽  
Author(s):  
N. BENKERROUM ◽  
Y. BOUHLAL ◽  
A. EL ATTAR ◽  
A. MARHABEN
Keyword(s):  
Escherichia Coli ◽  
Meat Products ◽  
Latex Agglutination ◽  
Agglutination Test ◽  
Escherichia Coli O157 ◽  
Selective Enrichment ◽  
E Coli ◽  
Meat Samples ◽  
Antigen Antibody ◽  
The City

Samples of meat and dairy products taken from the city of Rabat, Morocco, were examined for the presence of Escherichia coli O157 by the selective enrichment procedure followed by plating on cefixime–tellurite–sorbitol MacConkey agar and a latex agglutination test. The ability of isolates to produce Shiga toxins (ST1 or ST2) was also tested by an agglutination test using sensitized latex. Dairy samples (n = 44) included different products commonly consumed in the country. Meat samples (n = 36) were taken from traditional butchers because these products are generally marketed in this way. Random samples were taken from each product during the period of January through May. Of the 80 samples tested, 8 (10%) harbored E. coli O157. Four dairy and four meat samples were contaminated (9.1 and 11.1%, respectively). Of 10 E. coli O157 isolates from contaminated samples demonstrating true antigen-antibody agglutination, 5 (50%) produced either ST2 alone or ST2 plus ST1. Four of the five strains (80%) were meat isolates and produced ST2 with or without ST1, and the fifth was a dairy isolate producing ST2.

Effects of Recovery, Plating, and Inoculation Methods on Quantification of Escherichia coli O157:H7 and Listeria monocytogenes from Strawberries†

2004 ◽  
Vol 67 (11) ◽  
pp. 2436-2442 ◽  
Author(s):  
Y. HAN ◽  
R. H. LINTON ◽  
P. E. NELSON
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Storage Time ◽  
Storage Period ◽  
Escherichia Coli O157 ◽  
Inoculation Methods ◽  
E Coli ◽  
Agar Layer ◽  
Selective Plating ◽  
Phosphate Buffered Saline

Effects of different recovery and inoculation methods on quantification of Escherichia coli O157:H7 and Listeria monocytogenes from strawberries were studied. Strawberries were spot or dip inoculated with 7 to 8 log CFU per strawberry of each pathogen, air dried for 2 h, and stored for 1, 3, and 7 days at 4°C. The inoculated samples were stomached or washed with phosphate-buffered saline (PBS; pH 7.2) or with modified PBS (pH 8.4). Bacterial levels were determined using a direct selective plating, thin agar layer plating, or membrane-transferring plating (MTP) with tryptic soy agar and sorbital MacConkey agar (E. coli O157:H7) or modified Oxford agar (L. monocytogenes). Under most test conditions, washing with PBS followed by MTP had significantly higher (P < 0.05) recovery for both bacteria compared with other tested methods. Within a 7-day storage period for spot-inoculated strawberries, a stomaching step resulted in an injury of 0.9 to 1.4 log CFU for E. coli O157: H7 and 1.4 to 1.7 log CFU for L. monocytogenes. When a washing step was used instead, this resulted in an injury of only 0.2 to 0.6 log CFU for E. coli O157:H7 and 0.2 to 0.7 log CFU for L. monocytogenes. Both bacteria could survive on strawberry surfaces, but their recovered levels decreased with the increase of storage time at 4°C for both spot and dip inoculation methods. Dip inoculation generally had a lower recovery than spot inoculation. An ideal protocol to recover and enumerate E. coli O157:H7 and L. monocytogenes from strawberries involved shaking and washing samples with 100 ml of PBS for 15 min at 22°C coupled with a MTP enumeration method.

Effectiveness of Bacteriophages in Reducing Escherichia coli O157:H7 on Fresh-Cut Cantaloupes and Lettuce†

2009 ◽  
Vol 72 (7) ◽  
pp. 1481-1485 ◽  
Author(s):  
MANAN SHARMA ◽  
JITENDRA R. PATEL ◽  
WILLIAM S. CONWAY ◽  
SEAN FERGUSON ◽  
ALEXANDER SULAKVELIDZE
Keyword(s):  
Escherichia Coli ◽  
Foodborne Illness ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Iceberg Lettuce ◽  
Fresh Cut ◽  
Phosphate Buffered Saline

Consumption of produce contaminated with Escherichia coli O157:H7 has resulted in cases of foodborne illness. We determined the efficacy of a mixture of three E. coli O157:H7–specific bacteriophages (ECP-100) in reducing the number of viable E. coli O157:H7 on contaminated fresh-cut iceberg lettuce and cantaloupe. E. coli O157:H7 was spot inoculated on lettuce pieces (9 cm2) with a population of 3.76 log CFU/cm2, allowed to dry, and then sprayed with a control (phosphate-buffered saline) or ECP-100 to deliver 7.98 log PFU/cm2 to lettuce stored for 2 days at 4°C. Cut pieces of cantaloupe were spot inoculated with E. coli O157:H7 (4.55 log CFU/ml) and treated with the control or ECP-100 (6.69 log PFU/ml), and then stored at 4 or 20°C for up to 7 days. On days 0, 2, 5, and 7, cantaloupe samples were homogenized, and populations of E. coli O157:H7 were enumerated. Populations of E. coli O157:H7 on lettuce treated with ECP-100 on 0, 1, and 2 days (0.72, <0.22, and 0.58 log CFU/cm2 of lettuce) and stored at 4°C were significantly (P < 0.05) lower than those treated with the control (2.64, 1.79, and 2.22 log CFU/cm2), respectively. Populations on cut cantaloupes treated with ECP-100 on days 2, 5, and 7 (0.77, 1.28, and 0.96 log CFU/ml) and stored at 4°C were significantly lower than those cut cantaloupes treated with the control (3.34, 3.23, and 4.09 log CFU/ml), respectively. This study is the first to show the effectiveness of bacteriophages to reduce E. coli O157:H7 on fresh-cut lettuce and cantaloupes.

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