Escherichia coli (E.coli)
K.Theivendirarajah,
B.Sc(
Escherichia coli commonly known as E.coli is well known to microbiologist, geneticist, biochemist, biotechnologist, food technologist, public health and other medical professionals for various reasons. Off and on, it also appears in the media and gets the public attention as a dreaded organism causing sever illness through water and food contaminations. This article presents an overview of this organism.
The generic name - Escherichia is named after the German bacteriologist Theodor Escherichia who first isolated this organism and the species name - coli refer to that of colon or the large intestine which is its normal habitat. Like other bacteria present in the intestine E.coli also helps the body in the digestion process as well as in the absorption of vitamins.
E.coli is commonly isolated from intestines of humans and animals. Because E.coli is ubiquitous in feces, the presence of E.coli in water is considered to be an indicator of fecal contamination and the possible presence of enteric pathogens.
E.coli is Gram-negative, small straight rod(1-1.5 micron X 2-6 micron ), occurring singly or in pairs, motile by peritrichous flagella or non-motile, facultative anaerobic, both respiratory and fermentative. Glucose and other carbohydrates including lactose are readily fermented. Its saline features are that it is oxidase negative, citrate negative and indole positive. It belongs to the family Enterobacteriaceae and to a group called the coliforms.
E.coli is part of the bowel flora of healthy individual. However certain strains may cause intestinal and extra intestinal infections in imuno compromised as well as healthy individuals. Urinary track infections, bacteremia, meningitis and diarrheal diseases are the most common clinical syndromes caused by few pathogenic clones of E.coli. These are high-grade pathogens that require only a low inoculum to cause infections. Therefore detection of these pathogens in food or water prior to consumption is essential to prevent disease outbreaks.
There are four categories of diarrheagenic E.coli – (I) STEC Shiga toxin producing also referred to as enterohemorrhegic E.coli, (EHEC). (ii) enterotoxigenic(ETEC), (iii) enteropathogenic(EPEC), and (iv) enteroinvasive E.coli (EIEC).
E.coli 0157:H7 and other STEC serotypes cause illness in humans from a mild non bloody diarrhea to severe bloody diarrhoea - hemorrhagic colitis and haemolytic-uremic syndrome. These produce one or more shiga toxins also referred to as verocytotoxins. 0157 STEC is also capable to colonise dairy and beef cattle. For this reason you find 0157 STEC outbreaks from ground beef and other beef products. Other known vehicles of transmission include raw milk, sausage, roast beef, unchlorinated municipal water, raw vegetables, salads and mayonnaise.
The Wakerton tragedy that happened in
Recently in June 2003, an outbreak of food
poisoning due to E.coli 0157:H7
happened in a
E.coli strains including E.coli 0157:H7 can grow well in MacConkey agar. They ferment lactose present in the medium and appear deep pink to brick red on MAC plates. However since E.coli 0157 also ferments lactose, it cannot be distinguished from other E.coli on normal MAC plates. Most 0157 STEC strains do not ferment D-Sorbitol, hence sorbitol MAC (SMAC) plates are often used by medical labs for selective isolation of 0157 STEC strains. These will appear as colourless colonies on SMAC. They are further tested with 0157 antigens or latex reagents to identify the strain. Another selective plating media contains 4-methylumbelliferyl beta-D-glucoronide(MUG). Unlike other E.coli strains 0157 STEC strains are MUG negative. Confirmation of E.coli 0157:H7 requires identification of H7 flagella antigen. H7 specific antiserum is commercially available and is used to identify the strain.
Virulence testing of the enterotoxin producing E.coli can be performed by bio-assay methods .( e.g., cell culture or in vivo testing or the detection of gene sequence by DNA based methods such as polymerase chain reaction or colony blot hybridization techniques).
For the detection of E.coli in foods and other products, 10g or 25g of the product is suspended in 90 ml or 225 ml enrichment broth (e.g., Tryptic soya broth) and incubated at 30-35oC for 24-48 hours. Then a loopful from the broth is streaked on to MAC plate and incubated for 48 hours at 30-35oC. E.coli appears red with or without bile precipitate. The suspected colonies can be identified by streaking to Eosine methylene blue (EMB) agar plates. E.coli appears as blue black nucleated colonies with a green metallic sheen. Futher confirmation is done by testing on Enterotube II or API20E test systems. These test kits are available commercially.
Instead of using the normal 15 X 150 mm Petri plates of media for the isolation of E.coli, media coated on thin films called Petri films are nowadays used in some labs. Undiluted or diluted samples are added directly to plates at the rate of 1-5 ml per plate. Petri films contain dry media and a cold water soluble gelling agent.
Several new methods are now available to detect E.coli 0157 in foods. These methods use special enrichment broths and selective plates. Some use enzyme immunoassay and others use PCR technology to amplify specific DNA sequence unique to E.coli 0157:H7. For these new methods refer www.hc-sc.gc.ca/food-aliment/mh-dm/mhe-dme/compendium.
E.coli in water can be detected and enumerated by a number of simple methods. The two widely used methods are ; (1). The most probable number (MPN) method and (2) the membrane filter method. In the MPN method different volumes of water, 10ml, 1ml and 0.1ml are introduced into either lauryl sulphate broth tubes or lactose tubes (5 tubes per dilution).Gas production in the tubes at 48 hours is considered presumptive positive for coliforms. Confirmation is done by testing the positive tubes on Brilliant green bile broth and on EC tubes. EC positives are fecal coliforms and E.coli is one of them. E.coli can be further identified by API20E test or by using EC-MUG medium.
In the membrane filter method a known volume of water e.g. 100 ml is filtered through 0.45 micron filter which retains the bacteria present. The membrane is then placed on either an EMB or a mENDO agar plate. E.coli will appear as blue black colonies with a green metallic sheen on EMB and as red colonies with a metallic golden sheen on mENDO plates.
E.coli is the best studied organism for its biochemical and genetic properties. The research carried out on E.coli provided the meaning of life in molecular terms. The blue print of all living organisms, including man is stored in the wonder chemical compound DNA. The molecular structure and all properties associated with DNA such as its replication (reproduction), mutation, genetic recombination, DNA damage and repair mechanisms, genetic codes, gene structure, function, regulations and many others were elucidated by the research done on E.coli.
The discovery of restriction enzymes in E.coli paved the way for a whole lot of genetic manipulations and genetic engineering feats in biology. Many useful genetically modified organisms have been made using these techniques. The original restriction enzyme obtained from E.coli referred to as EcoR1 is still being used in many genetic manipulations. These advances made in E.coli provided the necessary tools for the successful mapping of the human genome, once thought to be an impossible task.
Jacob and Monod are two of the many 20th century biologists who earned their Nobel Prizes, a coveted trophy for a scientist, by working with E.coli. They worked on the lactose operon of E.coli and for the first time in 1961 showed that genes themselves control and regulate the activities of other genes. Today scientists try to block these regulator and operator sites to switch off mutated or bad genes to cure diseases. E.coli may be one of the most distant ancestors of man, yet it has the same genetic codes, reading frame, and speaks the same language as us in molecular terms in the expression of life.