scholarly journals A PCR Specific for Escherichia coli O157 Based on the rfb Locus Encoding O157 Lipopolysaccharide

1998 ◽  
Vol 36 (6) ◽  
pp. 1801-1804 ◽  
Author(s):  
Patricia M. Desmarchelier ◽  
Sima S. Bilge ◽  
Narelle Fegan ◽  
Leanne Mills ◽  
James C. Vary ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Extraction ◽  
Raw Milk ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Cell Lysates ◽  
O Antigen ◽  
Pcr Product

A PCR was developed for the detection of Escherichia coli O157 based on the rfbE O-antigen synthesis genes. A 479-bp PCR product was amplified specifically from E. coli O157 in cell lysates containing 200 or 2 CFU following crude DNA extraction. The PCR detected <1 CFU ofE. coli O157 per ml in raw milk following enrichment.

Polymerase Chain Reaction Assay for Detection of Escherichia coli O157:H7 and Escherichia coli O157:H−

2002 ◽  
Vol 65 (1) ◽  
pp. 5-11 ◽  
Author(s):  
TAKAHISA MIYAMOTO ◽  
NATSUKO ICHIOKA ◽  
CHIE SASAKI ◽  
HIROSHI KOBAYASHI ◽  
KEN-ICHI HONJOH ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Dna Sequence ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
Pcr Assay ◽  
E Coli ◽  
Pcr Product ◽  
Genomic Dnas ◽  
Polymerase Chain

The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non–E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H− and E. coli O157:H7. Since the DNA sequence from base 15 to base 1008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H− and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 × 100 to 3.5 × 102 CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42°C for 18 h and for the conventional cultural method.

Behavior of Escherichia coli O157:H7 during the Manufacture and Aging of Gouda and Stirred-Curd Cheddar Cheeses Manufactured from Raw Milk

2010 ◽  
Vol 73 (12) ◽  
pp. 2217-2224 ◽  
Author(s):  
DENNIS J. D'AMICO ◽  
MARC J. DRUART ◽  
CATHERINE W. DONNELLY
Keyword(s):  
Escherichia Coli ◽  
Population Level ◽  
Raw Milk ◽  
Escherichia Coli O157 ◽  
Milk Whey ◽  
Selective Enrichment ◽  
Point Counts ◽  
E Coli ◽  
Unpasteurized Milk ◽  
Low Levels

This study was conducted to examine the fate of Escherichia coli O157:H7 during the manufacture and aging of Gouda and stirred-curd Cheddar cheeses made from raw milk. Cheeses were manufactured from unpasteurized milk experimentally contaminated with one of three strains of E. coli O157:H7 at an approximate population level of 20 CFU/ml. Samples of milk, whey, curd, and cheese were collected for enumeration of bacteria throughout the manufacturing and aging process. Overall, bacterial counts in both cheese types increased almost 10-fold from initial inoculation levels in milk to approximately 145 CFU/g found in cheeses on day 1. From this point, counts dropped significantly over 60 days to mean levels of 25 and 5 CFU/g in Cheddar and Gouda, respectively. Levels of E. coli O157:H7 fell and stayed below 5 CFU/g after an average of 94 and 108 days in Gouda and Cheddar, respectively, yet remained detectable after selective enrichment for more than 270 days in both cheese types. Changes in pathogen levels observed throughout manufacture and aging did not significantly differ by cheese type. In agreement with results of previous studies, our results suggest that the 60-day aging requirement alone is insufficient to completely eliminate levels of viable E. coli O157:H7 in Gouda or stirred-curd Cheddar cheese manufactured from raw milk contaminated with low levels of this pathogen.

scholarly journals First isolation of Escherichia coli O157:H7 from faecal and milk specimens from Anatolian water buffaloes (Bubalus bubalus) in Turkey

2008 ◽  
Vol 79 (4) ◽  
Author(s):  
E. Seker ◽  
H. Yardimci
Keyword(s):  
Escherichia Coli ◽  
Raw Milk ◽  
Escherichia Coli O157 ◽  
Water Buffaloes ◽  
E Coli ◽  
Milk Samples ◽  
Cultural Methods ◽  
Faecal Samples

Three hundred rectal faecal samples and 213 raw milk samples obtained from the tanks and containers were examined using standard cultural methods. Escherichia coli O157:H7 was isolated from 11 (3.7 %) of 300 faecal samples and 3 (1.4 %) of 213 raw milk samples. It was determined that 8 (73 %) of E. coli O157:H7 strains isolated from faecal samples originated from water buffaloes younger than 2 years of age and 3 (27 %) from 2-year-old and older water buffaloes. This is the 1st isolation of Escherichia coli O157:H7 from faecal and milk samples of water buffaloes in Turkey.

Enumeration of Escherichia coli O157 in Outbreak-Associated Gouda Cheese Made with Raw Milk

2015 ◽  
Vol 78 (9) ◽  
pp. 1733-1737 ◽  
Author(s):  
ALEXANDER GILL ◽  
DENISE OUDIT
Keyword(s):  
Escherichia Coli ◽  
Raw Milk ◽  
Most Probable Number ◽  
Prolonged Storage ◽  
Escherichia Coli O157 ◽  
Probable Number ◽  
E Coli ◽  
Aging Period ◽  
Gouda Cheese ◽  
Cheese Products

In this article, we discuss the enumerative analysis for Escherichia coli O157 in two raw milk Gouda cheese products (A and B), implicated in an outbreak of 29 cases of E. coli O157:H7 illness that occurred across Canada in 2013. Samples were enumerated for E. coli O157 by most probable number (MPN) over a period of 30 to 60 days after the end of the outbreak. Samples (55.55 g) of product A (n = 14) were analyzed at 146 to 180 days postproduction. E. coli O157 was isolated from six samples at 19.9 to 44.6 MPN/kg. The E. coli O157 concentration of product A estimated from the results of all 14 samples was 9.5 MPN/kg. Samples (55.55 g) of product B (n = 20) were analyzed at 133 to 149 days postproduction. E. coli O157 was isolated from four samples at 19.9 MPN/kg. The E. coli O157 concentration of product B estimated from the results of all 20 samples was 3.7 MPN/kg. Analysis of a 305-g sample of product A (n = 1) stored at 4°C until 306 days postproduction revealed that the E. coli O157 concentration had declined to 3.6 MPN/kg. E. coli O157 could not be isolated from 555-g samples of product B (n = 5) after 280 days postproduction. The physicochemical parameters (pH, water activity, percent moisture, and percent salt) of both cheese products were found to be in the normal range for this type of product. The results of this study demonstrate that E. coli O157 could not replicate during storage at 4°C in the products tested but was capable of survival following aging and prolonged storage. This indicates that, if contaminated, the minimum 60-day aging period, which is required for raw milk Gouda cheeses, is not sufficient in all cases to ensure that the product does not contain viable cells of E. coli O157. The results also indicate that samples sizes greater than 100 g may be required to reliably detect E. coli O157 in cheese products associated with outbreaks.

Outbreak of Escherichia coli O157:H7 Infections Linked to Aged Raw Milk Gouda Cheese, Canada, 2013

2017 ◽  
Vol 81 (2) ◽  
pp. 325-331 ◽  
Author(s):  
Andrea Currie ◽  
Eleni Galanis ◽  
Pedro A. Chacon ◽  
Regan Murray ◽  
Lynn Wilcott ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Primary Source ◽  
Raw Milk ◽  
Core Sample ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Aging Period ◽  
Dairy Plant ◽  
Gouda Cheese ◽  
Canadian Provinces

ABSTRACT Between 12 July and 29 September 2013, 29 individuals in five Canadian provinces became ill following infection with the same strain of Escherichia coli O157:H7 as defined by molecular typing results. Five case patients were hospitalized, and one died. Twenty-six case patients (90%) reported eating Gouda cheese originating from a dairy plant in British Columbia. All of the 22 case patients with sufficient product details available reported consuming Gouda cheese made with raw milk; this cheese had been produced between March and July 2013 and was aged for a minimum of 60 days. The outbreak strain was isolated from the implicated Gouda cheese, including one core sample obtained from an intact cheese wheel 83 days after production. The findings indicate that raw milk was the primary source of the E. coli O157:H7, which persisted through production and the minimum 60-day aging period. This outbreak is the third caused by E. coli O157:H7 traced to Gouda cheese made with raw milk in North America. These findings provide further evidence that a 60-day ripening period cannot ensure die-off of pathogens that might be present in raw milk Gouda cheese after production and have triggered an evaluation of processing conditions, physicochemical parameters, and options to mitigate the risk of E. coli O157:H7 infection associated with raw milk Gouda cheese produced in Canada.

scholarly journals Isolation and Antimicrobial Susceptibility Profile of Escherichia coli O157 : H7 from Raw Milk of Dairy Cattle in Holeta District, Central Ethiopia

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Ashenafi Ababu ◽  
Dereje Endashaw ◽  
Haben Fesseha
Keyword(s):  
Escherichia Coli ◽  
Dairy Cattle ◽  
Antimicrobial Susceptibility ◽  
Large Scale ◽  
Raw Milk ◽  
Herd Size ◽  
Escherichia Coli O157 ◽  
Cross Sectional ◽  
E Coli ◽  
Antimicrobial Susceptibility Profile

A cross-sectional study was conducted in small, medium, and large-scale dairy farms of Holeta district to isolate, identify, and antimicrobial susceptibility profile of Escherichia coli O157 : H7 in raw milk of dairy cattle. A total of 210 lactating cows were selected for raw milk samples, and 19% (40/210) were found to be positive for E. coli whereas 5.2% (11/210) were confirmed as E. coli O157 : H7 positive using the Escherichia coli O157 latex test. Accordingly, all E. coli was highly susceptible to Ciprofloxacin (100%), Gentamycin (100%), Oxytetracycline (100%), and Tetracycline (63.63%). Furthermore, the resistance of 72.73%, 54.54%, 54.54%, and 45.45% was developed to Cefoxitin, Sulphamethoxazole, Cloxacillin, and Streptomycin, respectively. Factors such as parity, age, body condition, herd size, milk yield, udder hygiene, and udder lesion showed a statistically significant ( p < 0.05 ) association with the occurrence of E. coli infection in dairy cattle. In conclusion, in this study, a higher prevalence of Escherichia coli O157 : H7 and its drug susceptibility profile is an alarm for the health of the public, and awareness creation to the farm owners and the community is recommended.

Role of Nutrient Limitation in the Competition between Pseudomonas fluorescens and Escherichia coli O157:H7

2007 ◽  
Vol 70 (7) ◽  
pp. 1739-1743 ◽  
Author(s):  
R. C. MCKELLAR
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Fluorescens ◽  
Nutrient Limitation ◽  
Foodborne Pathogens ◽  
Raw Milk ◽  
Inhibitory Effect ◽  
Nutrient Broth ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Spoilage Microflora

Competition between spoilage microorganisms and foodborne pathogens provides a potentially simple approach to limiting the growth of pathogens. A strain of Pseudomonas fluorescens isolated from raw milk repressed growth of Escherichia coli O157:H7 at 22°C in nutrient broth once the maximum population density of the pseudomonad had been reached (9.6 log CFU ml−1). The presence of iron in the growth medium and the parallel inhibitory effect of a siderophore-deficient mutant of P. fluorescens precluded iron limitation as the mechanism of action. Medium depleted by prior growth of P. fluorescens prevented the growth of E. coli, and this effect was reversed by the replenishment of the nutrient broth, its component fractions, or the addition of soy peptones but not peptones derived from milk protein. This is the first report of competition between spoilage microflora and foodborne pathogens in which the mechanism was clearly shown to be nutrient limitation. These results suggest possible improvements in biocontrol systems to prevent pathogen growth on foods.

Influence of Lactic Acid Bacteria on Survival of Escherichia coli O157:H7 in Inoculated Minas Cheese during Storage at 8.5°C

2001 ◽  
Vol 64 (8) ◽  
pp. 1151-1155 ◽  
Author(s):  
SUSANA M. I. SAAD ◽  
CÉZAR VANZIN ◽  
MARICÊ N. OLIVEIRA ◽  
BERNADETTE D. G. M. FRANCO
Keyword(s):  
Escherichia Coli ◽  
Hazard Analysis ◽  
Control Point ◽  
Raw Milk ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Critical Control Point ◽  
Plastic Bags ◽  
Test Kit ◽  
Sterile Plastic

Minas cheese is a typical Brazilian fresh cheese, manufactured by addition of rennin and CaCl2 to milk, followed by draining the curd. The intrinsic characteristics of this product make it favorable for growth of pathogens, including Escherichia coli O157:H7. The influence of the addition of a commercial mesophilic type O lactic culture to this product on the growth of this pathogen during storage at 8.5°C was evaluated. Eight different formulations of Minas cheese were manufactured using raw or pasteurized milk and with or without salt and lactic culture. Individual portions of each formulation were transferred to sterile plastic bags and inoculated with E. coli O157:H7 to yield ca. 103 or 106 CFU/g. After blending by hand massaging the bags, samples were stored at 8.5°C for up to 14 days. E. coli O157:H7 was counted after 1, 2, 7, and 14 days of storage using 3M Petrifilm Test Kit-HEC. Counts in samples without added lactic culture showed a 2-log increase in the first 24 h and remained constant during the following 14 days. Counts in samples with added lactic culture showed a 0.5-log increase in the first 24 h, followed by a decrease. These variations were statistically significant (P &lt; 0.05). No significant variations (P &gt; 0.05) were obtained for cheese samples manufactured with pasteurized or raw milk, with or without salt. Results indicate that the addition of type O lactic culture may be an additional safeguard to well-established good manufacturing practices and hazard analysis and critical control point programs in the control of growth of E. coli O157:H7 in Minas cheese.

scholarly journals Presence of Escherichia coli O157 and O157:H7 in raw milk and Van herby cheese

2015 ◽  
Vol 59 (4) ◽  
pp. 511-514 ◽  
Author(s):  
Yakup Can Sancak ◽  
Hakan Sancak ◽  
Ozgur Isleyici
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
Drug Administration ◽  
Shiga Toxin ◽  
Raw Milk ◽  
Escherichia Coli O157 ◽  
Human Pathogens ◽  
Emerging Pathogens ◽  
E Coli ◽  
Milk Samples

Abstract The Shiga toxin-producing Escherichia coli (STEC) strains are currently considered important emerging pathogens threatening public health. Among Shiga toxin-producing Escherichia coli, E. coli O157:H7 strains have emerged as important human pathogens. This study was conducted to determine the presence of Escherichia coli O157 and O157:H7 in raw milk samples and Van herby cheese samples. For this purpose, 100 samples of raw milk were collected and 100 samples of herby cheese sold for consumption in Van province in Turkey were obtained from grocers and markets in order to detect the presence of Escherichia coli O157 and O157:H7. The method of E. coli O157 and O157:H7 isolation proposed by the Food and Drug Administration (FDA) was used. E. coli O157 in raw milk and herby cheese samples was found in 11% and 6% of samples respectively, and E. coli O157:H7 was found in 2% of herby cheese samples. No E. coli O157:H7 was detected in raw milk samples. This study showed that raw milk was contaminated with E. coli O157 and herby cheese was contaminated with both E. coli O157 and E. coli O157:H7; therefore, herby cheese poses a serious risk to public health.

Evaluation of the VIDAS Methodology for Detection of Escherichia coli O157 in Food Samples

1998 ◽  
Vol 61 (7) ◽  
pp. 917-920 ◽  
Author(s):  
C. VERNOZY-ROZAND ◽  
C. MAZUY ◽  
S. RAY-GUENIOT ◽  
S. BOUTRAND-LOEÏ ◽  
A. MEYRAND ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Raw Milk ◽  
Immunomagnetic Separation ◽  
Food Samples ◽  
Positive Sample ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Contaminated Food ◽  
Capture And Release ◽  
Retail Food

An automated enzyme-linked fluorescence immunoassay (ELFA), the VIDAS E. coli O157 method, was compared with immunomagnetic separation (IMS) followed by culture on cefixime tellurite sorbitol MacConkey agar (CT-SMAC) for detecting Escherichia coli O157 in artificially and naturally contaminated food samples including raw milk cheeses, poultry, raw sausages, and ground beef retail samples. Confirmation of the samples positive according to the ELFA was performed by use of an automated immunoconcentration system, VIDAS ICE, which allows selective capture and release of target organisms. A total of 496 retail food samples were examined. Seventeen food samples gave positive values with the ELFA method, and among them 9 food samples were confirmed by the ICE method. Eight were shown to contain sorbitol-positive, O157-positive, H7-negative, motile, non-verotoxin-producing E. coli. The ninth positive sample contained an O157-positive, H7-negative, sorbitol-negative, non-verotoxin-producing E. coli. The IMS technique only allowed confirmation of this sorbitol-negative, non-verotoxin-producing E. coli O157.

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