Escherichia coli are Gram negative rod-shaped organisms found naturally in the lower intestines of warm blooded organisms. Most serotypes of this organism are relatively harmless, making up a small percentage of bacterial colonization in the gut. These serotypes prevent the establishment of pathogenic strains.
E. coli is one of the most common bacteria found in the gut of animals. This includes humans. There are other animal species that contain this organism in the gut including reptiles and fish. E. coli colonizes the gut and can cause infection in the urinary tract and brain stem (meningitis) as well as intestinal diseases referred to as gastroenteritis. There are five classes of E. coli that produce disease. The most serious disease is the Enterohemorrhagic (EHEC) class. These organisms can cause diarrhea distinct from some others (including Shigella) in that there is copious bloody discharge and no fever. The life threatening situation is its toxic effects on the kidneys (hemolytic uremia).
Why Test for the Presence of E. coli?
E. coli is often used as indicator organisms to test the effectiveness of effluent disinfection in a wastewater treatment plant, on animal products as well as in nutraceutical and pharmaceutical products. While these organisms are generally harmless, they do live under the same conditions that human pathogens live. Since we cannot test for every pathogen, we test for easily detectable indicator organisms. The assumption is that if we kill the indicator organisms then we most likely kill the pathogens during effluent disinfection. E. coli has reemerged as an indicator, partly facilitated by the introduction of newer methods that can rapidly identify E. coli.
Current Methodology
The current methodology can take anywhere from 3-7 days and includes multiple broths, agars, transfers and temperatures. Different methodologies are utilized by the various industries. Below are some examples:
Nutraceutical and Dietary supplements: The protocol described in USP <2022> requires absence of the organisms in 10 grams of product. A 1:10 dilution of product is made into TSB or another enrichment medium. This mixture is incubated for 24 to 48 hours at 30°C to 35°C, and then 1.0 mL is transferred to 10 mL of MacConkey broth. This mixture is incubated for 24 to 48 hours at 42°C to 44°C. A loop from the MacConkey broth is transferred to MacConkey agar and the plate is incubated for 18 to 24 hours at 30°C to 35°C. If typical colonies appear, these colonies are then transferred to Levine Eosin Methylene Blue agar and incubated 24 to 48 hours at 30°C to 35°C. If none of the colonies exhibit green metallic sheen under reflected light or if none of the colonies exhibit a blue-black appearance under transmitted light, the sample meets the requirement for the absence of Escherichia coli. Because results can sometimes be misread due to interpretation of plates, identification may be run on the sample(s) adding another 2-3 days of testing.
Pharmaceutical: The protocol described in USP <62> again requires the absence of E. coli in 10 grams of product. A 1:10 dilution is made and incubated for 18 to 24 hours at 30°C to 35°C. From this mixture, 1.0 mL is transferred to 100 mL of MacConkey broth and incubated 24 to 48 hours at 42°C to 44°C. A loop is transferred to MacConkey agar and this plate is incubated 18 to 72 hours at 30°C to 35°C. Growth of typical colonies indicates the presence of E. coli which is confirmed with an identification test.
Food Testing: The food industry follows Bacteriological Analytical Manual (BAM). In most cases, the Most Probable Number (MPN) method is utilized. This is labor intensive, multi-step assay consists of presumptive (in LST tubes), confirmed (in BGLB tubes), and completed phases (in EC tubes). In the assay, serial dilutions of a sample are inoculated into broth media. Analysts score the number of gas positive (fermentation of lactose) tubes, from which the other 2 phases of the assay are performed and then uses the combinations of positive results to consult a statistical table. From this table, the analyst is able to estimate the number of organisms present. The 3-tube MPN test is used for testing most foods. The 5-tube MPN is used for water, shellfish and shellfish harvest water testing, and there is also a 10-tube MPN method that is used to test bottled water or samples that are not expected to be highly contaminated. Positive EC tubes must be transferred to L-EMB agar and if typical colonies are observed, they must be further identified.
The BioLumix E. coli Vial
The E. coli vial, or EC vial, is a membrane vial that is monitor by the fluorescent signal in the instrument. The membrane separates the incubation zone from the reading zone. If E. coli is present, it utilizes MUG (4-Methylumbelliferyl-3-D-Glucuronide) through an enzymatic reaction to create fluorescence. A 1:10 dilution is made in TSB or a similar growth medium and this mixture is incubated for 18-24 hours at 35°C. From the TSB dilution, 0.1 mL is added to an EC vial containing the MUG supplement and then tested in the BioLumix instrument. The EC assay runs for 18 hours in the instrument. An example of E. coli curves is shown in the graph. If detection occurs, a confirmation test is performed. From the time the sample is prepared to the time the confirmation is complete, the EC test takes only 2 days, saving valuable time.
The BioLumix assay is much simpler to perform, requiring less labor and disposables as any to the standard method. It is faster (completed in 30 hours), accurate, automated, and it eliminates any product interference due to the two zone vial.
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