In 1885, Theodor Escherich first isolated Bacterium coli from children's fecal samples. After the 1920s, this bacterium underwent a name change and became known as Escherichia coli. There were growing suspicions that these bacteria were responsible for sporadic cases of gastroenteritis with a notable mortality rate in children. (1)
By 1940, E. coli had been confirmed as an enteric pathogen, prompting the establishment of measures to control its spread in developed countries. The food industry also recognized the significance of this bacterium. Since the early 20th century, it has served as an indicator of fecal contamination in both water and food sources. (1)
The majority of E. coli strains are typically harmless to their hosts. However, it is important to note that there are certain strains that can be pathogenic to humans. The most prevalent pathogenic strains are commonly classified into two groups: E. coli Shiga-Toxin (STEC), including E. coli enterohemorrhagic (EHEC), which encompasses E. coli O157:H7, and E. coli enteropathogenic (EPEC). These classifications are based on their virulence factors, pathogenicity mechanisms, clinical syndromes, and serological characteristics. It's worth mentioning that there are other E. coli strains with distinct properties beyond these two common categories.
E. coli bacteria are gram—negative bacteria that belong to the Enterobacteriaceae family. The cells are rod-shaped and can be either motile (by flagella) or immotile. E. coli strains can be differentiated based on somatic antigen (O), flagellar antigen (H) or capsular antigen (K). Presence of fimbriae and other related structures play a big part in bacterial virulence. (1)
Regarding growth conditions, E. coli strains thrive in a temperature range of 7 to 46ºC, with an optimal growth temperature of 37ºC. Pathogenic strains can endure refrigeration temperatures for up to 1-5 weeks. The pH affects E. coli growth differently depending on the type of acid; it can grow at pH 4.5 when adjusted with chloridic acid but not when adjusted with lactic acid. Pathogenic strains cannot grow in cheese with a pH below 5.4. The minimum water activity for E. coli growth is 0.95, with growth possible up to 6.5% NaCl concentration but inhibited above 8.5%. Lastly, E. coli is facultative anaerobic, meaning it grows with or without oxygen. It’s susceptible to destruction by radiation with oxygen intensifying its lethal effect, especially at 45-55ºC.
STEC/ VTEC and EHEC
E. coli Shiga-Toxin (STEC) or E. coli Verotoxigenic (VTEC) are strains of Escherichia coli bacteria that produce the Shiga toxin (or verotoxin) (Vtx), encoded by vtx1 or vtx2 genes, (also known as Shiga-like toxins – Stx – corresponding to stx1 and stx2 genes). These toxins can cause damage to the lining of the intestines, leading to symptoms like bloody diarrhea.
E. coli enterohaemorrhagic (EHEC) is a subset of STEC/VTEC, which includes E. coli O157:H7. This strain, in addition to the stx-encoding genes, such as stx1 and stx2, usually carry the attaching and effacing gene (eae; intimin-coding). E. coli enterohaemorrhagic (EHEC), is recognized as one of the main pathogens of foodborne illness in humans. Is associated with watery diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome.
The strains are usually classified into various serogroups based on their O (somatic) and H (flagellar) antigens. The serogroups help categorize the different strains. Some of the common STEC/VTEC serogroups associated with human infections include O157 (e.g., E. coli O157:H7, one of the most well-known STEC serogroups and is associated with severe illnesses), O26, O45, O55, O103, O111 (cause similar diseases to O157 strains), O117, O121, O145 and O91.
The emphasis on E. coli O157 as opposed to other serotypes has been further reinforced due to its ease of isolation using culture methods. In contrast, the isolation of other serotypes has been sub-diagnosed by the lack of suitable methods to isolate those strains, relying instead on PCR-based techniques.
E. coli Enteropathogenic (EPEC) was the first recognized pathogenic group, and presently, continues to be a leading cause of diarrhoea among infants from developing countries worldwide.
EPEC produces an outer membrane protein called intimin, which is a virulence factor (adhesin) encoded by eae gene. This protein plays a critical role mediating the attachment of EPEC to intestinal cells. By definition, all EPEC do not have stx genes and are all uniformly eae positive (+). Additionally, the Locus of Enterocyte Effacement (LEE) is a genetic region within EPEC’s genome that is essential for its pathogenicity. LEE contains genes responsible for the formation of specialized attachment and effacement lesion on the surface of intestinal cells. Leading to diarrheal disease in infected individuals.
EPEC is classified into different serogroups based on the presence of specific O (somatic) and H (flagellar) antigens. These serogroups help distinguish different strains of EPEC. Some of the common EPEC serogroups include O26, O55¸ O86, O111, O114, O119, O125, O126, O127, O128, O142 and O158.
Main sources of contamination
The main habitat of E. coli is the intestinal tract of humans and other hot-blooded animals. Transmission of infections caused by E. coli follow three pathways: direct contact with animals, human contact, and consumption of contaminated food. The different methods of transmission in the food industry are:
- Oral-fecal during animal husbandry.
- Land contamination – when animal stool is used as fertilizer without previous treatment.
- Fecal contamination of carcasses – ignoring good practices of slaughtering.
- Contaminated raw milk consumption.
- Contaminated milk consumption from cows with mastitis caused by E. coli.
- Contaminated water consumption. (1)
Contaminated Water Sources and Food-Related E. coli Infections:
Human and animal waste can pollute various water sources, including ground and surface waters like streams, rivers, lakes, and water used for crop irrigation. While public water systems use methods like chlorine, ultraviolet light, or ozone to eliminate E. coli, some outbreaks have still been linked to polluted municipal water supplies. Private water wells, especially in rural areas, pose a greater concern due to the lack of effective disinfection methods. Additionally, documented cases of E. coli infections have occurred in individuals who have come into contact with pools or lakes contaminated with fecal matter.
On the other hand, irrigation water contaminated by sewage and individuals, including both animals and operators, infected with E. coli ETEC, can also become pathways for food contamination. Notably, in countries with strict hygiene standards, ETEC is not a significant public health concern.
Concerning contaminated food, there have been numerous outbreaks and isolated cases in recent decades, drawing public attention due to their severity and high mortality rates. These incidents are often attributed to water or food contamination with fecal matter, primarily resulting from inadequate sanitation, poor manufacturing practices, and insufficient personal hygiene. Foods most frequently associated with E. coli infections include undercooked beef products, such as hamburgers, cured sausages, alfalfa seeds, lettuce, non-pasteurized fruit juices, cured cheese, and raw milk.
The establishment of codes of good practices with hygienic slaughtering practices and corrective actions with the objective of reducing fecal contamination along the food chain may contribute to reduce public health dangers associated with E. coli. (1)
Education in hygienic food handling for workers in farms, abattoirs, and the food production industry is essential to minimize microbiological contamination. The primary goal of contamination control is to reduce the presence of microorganisms during the breeding and slaughtering of animals, especially cattle. The most effective means of eliminating STEC from foods involves introducing bactericidal treatments such as heating (e.g., cooking or pasteurization) or irradiation. Infection prevention for E. coli entails strict adherence to temperature control measures throughout the cold chain and avoiding the consumption of undercooked beef, particularly bovine, as well as unpasteurized milk and untreated water. (1,2)
Furthermore, the implementation of auto-control systems such as HACCP along the chain is considered as very important prevention strategy.
Escherichia coli Detection
Detecting pathogenic E. coli can be challenging due to its genetic similarity to non-pathogenic strains and the need to identify specific virulence markers. The traditional method of growing E. coli in specific culture media is time-consuming and does not distinguish between all pathogenic strains.
The real-time PCR technique, on the other hand, is much more sensitive and allows specific DNA segments of pathogenic E. coli to be amplified. The primers used are designed to bind to virulence genes, such as the shiga toxin (stx) verocytoxins (vtx) and intimin (eae) genes.
BPMR presents 4 kits designed for E.coli. Below are some details of the kits:
- BPMR kits take full advantage of real-time PCR to enable a quick and reliable method for vegan food fraud detection.
- These kits are based on gene amplification and detection using real-time PCR with an DNA-based assay.
- Ready-to-use PCR reagents contain everything required to detect targeted organisms with high sensitivity and specificity.
- The SUPREME REAL TIME PCR DETECTION TEST KIT Escherichia coli and REAL TIME PCR DETECTION TEST KIT Escherichia coli are both kits that allow the detection of pathogenic E. coli associated with the pathotypes EPEC, STEC and the subgroup EHEC associated with the combination of the virulence genes stx1 and/or stx2 and eae.
- The SUPREME REAL TIME PCR DETECTION TEST KIT Escherichia coli is validated by AOAC INTERNATIONAL. A validation study was performed, as part of the AOAC Performance Tested Methods Program, that demonstrated no differences in results between the SUPREME REAL TIME PCR DETECTION TEST KIT Escherichia coli and reference methods.
- The SUPREME REAL TIME PCR DETECTION TEST KIT E. coli O157:H7 allows for the detection of pathogenic E. coli O157 and allows the simultaneous detection of the serotype O157:H7 DNA.
- The REAL TIME PCR DETECTION TEST KIT Escherichia Coli Serotype / Serogroup (O157, O26, O111, O103, And O145) allows detection of E. coli serotypes O157, O26, O111, O103, and O145 in food products, animal feedstuff and environmental samples.
- All kits include an internal control (IC) that allows the exclusion of false negative results.
- Our kits have incorporated a Hot Start polymerase to enable reliable PCR amplification. The mix offers highly reproducible DNA synthesis.
Various tests were performed to ensure both kit quality such as inclusivity, sensitivity, and exclusivity:
TEST KIT Escherichia coli - 100 % Inclusivity, determined in 18 positive samples for eae, vtx1 and/or vtx2 genes of Escherichia coli.
TEST KIT SUPREME Escherichia coli - For inclusivity, 66 E. coli strains containing stx1 and/or stx2 and eae genes (such as O157, O26, O1O3, O111, and O145) of 67 were correctly detected by the corresponding gene target. The samples used were mainly culture collections, proficiency tests and local isolates. Each strain were enriched in mTSB+N at 37 ± 1°C for 18–24 h per ISO/TS 1316:20125.
TEST KIT E. coli O157:H7 - 100 % Inclusivity, determined in 9 strains of E. coli O157:H7 and E. coli O157.
TEST KIT Escherichia coli serogroups - 100 % Inclusivity, determined in 45 Escherichia coli strains and 5 positive cultures.
TEST KIT Escherichia coli - A detection Limit of 1 to 10 cells per 25g of food sample can be achieved after enrichment. The REAL TIME DETECTION KIT E.coli EPEC, VTEC and EHEC detects down to 103-104 cfu/mL in enrichment cultures. This kit has a reaction sensitivity of 25 pg of target DNA.
TEST KIT SUPREME Escherichia coli - In the AOAC validation study, the SUPREME REAL TIME DETECTION KIT E. coli showed a comparable detection or performance to the reference method (ISO/TS 13136:20125) for the foods tested, raw ground beef, orange juice, salad (green, purple lettuce and coriander) and cream cheese.
TEST KIT E. coli O157:H7 - Target amplification was observed in samples with 1 to 10 Cells per 25g of food sample.
TEST KIT Escherichia coli - 100 % Exclusivity, determined using 13 strains of closely related organisms or occurring in the same habitat, including non-pathogenic E. coli and other serotypes of E. coli.
TEST KIT SUPREME Escherichia coli - 100 % Exclusivity, determined using 37 strains of closely related organisms or occurring in the same habitat.
TEST KIT Escherichia coli serogroups - 100 % Exclusivity, determined using 30 strains of closely related organisms or occurring in the same habitat.
- For DNA extraction, our BIOPREMIER DNA Extraction from Food kit allows efficient extraction and purification of DNA samples from challenging matrices containing high levels of polyphenols. Furthermore, the purified DNA products can be used directly on the PCR.
BPMR present to you a global solution from the extraction and purification of DNA from food sources for pathogen identification.
- Quick results (one-two days) in few steps: first Food Source DNA Purification, followed by Food Pathogen Testing and lastly Data Analysis. Check below the “A simple Three-step Protocol”. - Accessible and easy to use thanks to a ready-to-use kits.
- Harmonized protocols for Real Time PCR detection.
- Compatible in the most used real time PCR equipment’s.
- Includes Positive and Negative PCR controls and Internal control.
This flexible and sturdy protocol fits smoothly in any analytical lab workflow to reach the best productivity! Ranges from low to high throughput testing, with simple to use user interface, to, in the end, provide efficient sample interpretation.
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Real Time Pcr Detection Test Kit Escherichia Coli Supreme (Stx1, Stx2 And Eae Genes)
Real Time Pcr Detection Test Kit Supreme Escherichia Coli O157:H7 / O157 (Duplex)
Real Time Pcr Detection Test Kit Escherichia Coli Serotype / Serogroup (O157, O26, O111, O103, And O145)