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Friday, May 8, 2009

Escherichia coli


(Escherichia coli can generally cause several intestinal and extra-intestinal infections such as urinary tract infections, meningitis, peritonitis, mastitis, septicemia and pneumonia. E. coli is often the causative agent of Traveler's diarrhea. The primary source of infection is ingestion of fecally contaminated food or water. )


Escherichia coli (commonly E. coli, is a bacterium that is commonly found in the lower intestine of warm-blooded animals. Most E. coli strains are harmless, but some, such as serotype O157:H7, can cause serious food poisoning in humans, and are occasionally responsible for costly product recalls. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, or by preventing the establishment of pathogenic bacteria within the intestine.

E. coli are not always confined to the intestine, and their ability to survive for brief periods outside the body makes them an ideal indicator organism to test environmental samples for fecal contamination. The bacteria can also be grown easily and its genetics are comparatively simple and easily-manipulated, making it one of the best-studied prokaryotic model organisms, and an important species in biotechnology. E. coli was discovered by German pediatrician and bacteriologist Theodor Escherich in 1885, and is now classified as part of the Enterobacteriaceae family of gamma-proteobacteria.


Strains

(Model of successive binary fission in E. coli)

A strain of E. coli is a sub-group within the species that has unique characteristics that distinguish it from other E. coli strains. These differences are often detectable only on the molecular level; however, they may result in changes to the physiology or lifecycle of the bacterium. For example, a strain may gain pathogenic capacity, the ability to use a unique carbon source, the ability to inhabit a particular ecological niche or the ability to resist antimicrobial agents. Different strains of E. coli are often host-specific, making it possible to determine the source of fecal contamination in environmental samples. Depending on which E. coli strains are present in a water sample, for example, assumptions can be made about whether the contamination originated from a human, other mammal or bird source.

New strains of E. coli evolve through the natural biological process of mutation, and some strains develop traits that can be harmful to a host animal. Although virulent strains typically cause no more than a bout of diarrhea in healthy adult humans, particularly virulent strains, such as O157:H7 or O111:B4, can cause serious illness or death in the elderly, the very young or the immunocompromised.


Biology and biochemistry

(Escherichia coli cells propel themselves with flagella (long, thin structures) arranged as bundles that rotate counter-clockwise, generating torque to rotate the bacterium clockwise.)

E. coli is Gram-negative, facultative anaerobic and non-sporulating. The cells are about 2 micrometres (μm) long and 0.5 μm in diameter, with a cell volume of 0.6 - 0.7 μm3. It can live on a wide variety of substrates. E. coli uses mixed-acid fermentation in anaerobic conditions, producing lactate, succinate, ethanol, acetate and carbon dioxide. Since many pathways in mixed-acid fermentation produce hydrogen gas, these pathways require the levels of hydrogen to be low, as is the case when E. coli lives together with hydrogen-consuming organisms such as methanogens or sulfate-reducing bacteria.

Optimal growth of E. coli occurs at 37°C, but some laboratory strains can multiply at temperatures of up to 49°C. Growth can be driven by aerobic or anaerobic respiration, using a large variety of redox pairs, including the oxidation of pyruvic acid, formic acid, hydrogen and amino acids, and the reduction of substrates such as oxygen, nitrate, dimethyl sulfoxide and trimethylamine N-oxide.

Strains that possess flagella can swim and are motile, but other strains lack flagellum. The flagella of E. coli have a peritrichous arrangement.

E. coli and related bacteria possess the ability to transfer DNA via bacterial conjugation, transduction or transformation, which allows genetic material to spread horizontally through an existing population. This process led to the spread of the gene encoding shiga toxin from Shigella to E. coli O157:H7, carried by a bacteriophage.


Normal role

E. coli normally colonizes an infant's gastrointestinal tract within 40 hours of birth, arriving with food or water or with the individuals handling the child. In the bowel, it adheres to the mucus of the large intestine. It is the primary facultative organism of the human gastrointestinal tract. As long as these bacteria do not acquire genetic elements encoding for virulence factors, they remain benign commensals.


Role in disease

Virulent strains of E. coli can cause gastroenteritis, urinary tract infections, and neonatal meningitis. In rarer cases, virulent strains are also responsible for hæmolytic-uremic syndrome (HUS), peritonitis, mastitis, septicemia and Gram-negative pneumonia. Recently it is thought that E. coli and certain other foodborne illnesses can sometimes trigger serious health problems months or years after patients survived that initial bout.



Gastrointestinal infection

(Low-temperature electron micrograph of a cluster of E. coli bacteria, magnified 10,000 times. Each individual bacterium is oblong shaped.)

Certain strains of E. coli, such as O157:H7, O121 and O104:H21, produce toxins. Food poisoning caused by E. coli are usually associated with eating unwashed vegetables and meat contaminated post-slaughter. O157:H7 is further notorious for causing serious and even life-threatening complications like hemolytic-uremic syndrome (HUS). This particular strain is linked to the 2006 United States E. coli outbreak of fresh spinach. Severity of the illness varies considerably; it can be fatal, particularly to young children, the elderly or the immunocompromised, but is more often mild. E. coli can harbor both heat-stable and heat-labile enterotoxins. The latter, termed LT, contains one 'A' subunit and five 'B' subunits arranged into one holotoxin, and is highly similar in structure and function to Cholera toxins. The B subunits assist in adherence and entry of the toxin into host intestinal cells, while the A subunit is cleaved and prevents cells from absorbing water, causing diarrhea. LT is secreted by the Type 2 secretion pathway.

If E. coli bacteria escape the intestinal tract through a perforation (for example from an ulcer, a ruptured appendix, or a surgical error) and enter the abdomen, they usually cause peritonitis that can be fatal without prompt treatment. However, E. coli are extremely sensitive to such antibiotics as streptomycin or gentamicin. This could change since, as noted below, E. coli quickly acquires drug resistance. Recent research suggests that treatment with antibiotics does not improve the outcome of the disease, and may in fact significantly increase the chance of developing haemolytic uraemic syndrome.

Intestinal mucosa-associated E. coli are observed in increased numbers in the inflammatory bowel diseases, Crohn's disease and ulcerative colitis. Invasive strains of E. coli exist in high numbers in the inflamed tissue, and the number of bacteria in the inflamed regions correlates to the severity of the bowel inflammation.


Virulence properties

Enteric E. coli (EC) are classified on the basis of serological characteristics and virulence properties. Virotypes include:

  • Enterotoxigenic E. coli (ETEC): causative agent of diarrhea (without fever) in humans, pigs, sheep, goats, cattle, dogs, and horses. ETEC uses fimbrial adhesin s (projections from the bacterial cell surface) to bind enterocyte cells in the small intestine. ETEC can produce two proteinaceous enterotoxins: the larger of the two proteins, LT enterotoxin, is similar to cholera toxin in structure and function, while the smaller protein, ST enterotoxin causes cGMP accumulation in the target cells and a subsequent secretion of fluid and electrolytes into the intestinal lumen. ETEC strains are non-invasive, and they do not leave the intestinal lumen. ETEC is the leading bacterial cause of diarrhea in children in the developing world, as well as the most common cause of traveler's diarrhea. Each year, ETEC causes more than 200 million cases of diarrhea and 380,000 deat hs, mostly in children in developing countries.
  • Enteropathogenic E. coli (EPEC): causative agent of diarrhea in humans, rabbits, dogs, cats and horses. Like ETEC, EPEC also causes diarrhea, but the molecular mechanisms of colonization and etiology are different. EPEC lack fimbriae, ST and LT toxins, but they utilize an adhesin known as intimin to bind host intestinal cells. This virotype has an array of virulence factors that are similar to those found in Shigella, and may possess a shiga toxin. Adherence to the intestinal mucosa causes a rearrangement of actin in th e host cell, causing significant deformation. EPEC cells are moderately-invasive (i.e. they enter host cells) and elicit an inflammatory response. Changes in intestinal cell ultrastructure due to "attachment and effacement" is likely the prime cause of diarrhea in those afflicted with EPEC.
  • Enteroinvasive E. coli (EIEC): found only in humans. EIEC infection causes a syndrome that is identical to Shigellosis, with profuse diarrhea and high fever. EIE C are highly invasive, and they utilize adhesin proteins to bind to and enter intestinal cells. They produce no toxins, but severely damage the intestinal wall through mechanical cell destruction.
  • Enterohemorrhagic E. coli (EHEC): found in humans, cattle, and goats. The sole member of this virotype is strain O157:H7, which causes bloody diarrhea and no fever. EHEC can cause hemolytic-uremic syndrome and sudden kidney failure. It uses bacterial fimbriae for attachment, is moderately-invasive and possesses a p hage-encoded Shiga toxin that can elicit an intense inflammatory response.
  • Enteroaggregative E. coli (EAggEC): found only in humans. So named because they have fimbriae which aggregate tissue culture cells, EAggEC bind to the intestinal mucosa to cause watery diarrhea without fever. EAggEC are non-invasive. They produce a hemolysin and an ST enterotoxin similar to that of ETEC.

Epidemiology of gastrointestinal infection

Transmission of pathogenic E. coli often occurs via fecal-oral transmission. Common routes of transmission include: unhygienic food preparation, farm contamination due to manure fertilization, irrigation of crops with contaminated greywater or raw sewage, feral pigs on cropland, or direct consumption of sewage-contaminated water. Dairy and beef cattle are primary reservoirs of E. coli O157:H7, and they can carry it asymptomatically and shed it in their feces. Food products associated with E. coli outbreaks include raw ground beef, raw seed sprouts or spinach, raw milk, unpasteurized juice, and foods contaminated by infected food workers via fecal-oral route.

According to the U.S. Food and Drug Administration, the fecal-oral cycle of transmission can be disrupted by cooking food properly, preventing cross-contamination, instituting barriers such as gloves for food workers, instituting health care policies so food industry employees seek treatment when they are ill, pasteurization of juice or dairy products and proper hand washing requirements.

Shiga toxin-producing E. coli (STEC), specifically serotype O157:H7, have also been transmitted by flies, as well as direct contact with farm animals, petting zoo animals, and airborne particles found in animal-rearing environments.

((a) E. coli–GFP infection in the stomach immediately after gavage of 1011E. coli–GFP.
(b) E. coli–GFP infection in the small intestine 10 min after gavage.
(c) E. coli–GFP infection in the small intestine 20 min after gavage.
(d) E. coli–GFP infection in the small intestine 30 min after gavage.
(e) E. coli–GFP infection in the small intestine 40 min after gavage.
(f) E. coli–GFP infection in the small intestine 50 min after gavage.
(g) E. coli–GFP infection in the small intestine 60 min after gavage.
(h) E. coli–GFP infection in the colon 120 min after gavage.
(i) E. coli–GFP infection in the colon immediately after enema of 1011E. coli–GFP1.)

Urinary tract infection

Uropathogenic E. coli (UPEC) is responsible for approximately 90% of urinary tract infections (UTI) seen in individuals with ordinary anatomy. In ascending infections, fecal bacteria colonize the urethra and spread up the urinary tract to the bladder. Because women have a shorter urethra than men, they are 14-times more likely to suffer from an ascending UTI.

Uropathogenic E. coli utilize P fimbriae (pyelonephritis-associated pili) to bind urinary tract endothelial cells and colonize the bladder. These adhesins specifically bind D-galactose-D-galactose moieties on the P blood group antigen of erythrocytes and uroepithelial cells. Approximately 1% of the human population lacks this receptor, and its presence or absence dictates an individual's susceptibility to E. coli urinary tract infections. Uropathogenic E. coli produce alpha- and beta-hemolysins, which cause lysis of urinary tract cells.

UPEC can evade the body's innate immune defenses (e.g. the complement system) by invading superficial umbrella cells to form intracellular bacterial communities (IBCs). They also have the ability to form K antigen, capsular polysaccharides that contribute to biofilm formation. Biofilm-producing E. coli are recalcitrant to immune factors and antibiotic therapy and are often responsible for chronic urinary tract infections. K antigen-producing E. coli infections are commonly found in the upper urinary tract.

Descending infections, though relatively rare, occur when E. coli cells enter the upper urinary tract organs (kidneys, bladder or ureters) from the blood stream.


Laboratory diagnosis

In stool samples microscopy will show Gram negative rods, with no particular cell arrangement. Then, either MacConkey agar or EMB agar (or both) are inoculated with the stool. On MacConkey agar, deep red colonies are produced as the organism is lactose positive, and fermentation of this sugar will cause the medium's pH to drop, leading to darkening of the medium. Growth on Levine EMB agar produces black colonies with greenish-black metallic sheen. This is diagnosic of E. coli. The organism is also lysine positive, and grows on TSI slant with a (A/A/g+/H2S-) profile. Also, IMViC is ++-- for E. coli; as it's indol positive (red ring) and methyl red positive (bright red), but VP negative (no change-colorless) and citrate negative (no change-green color). Tests for toxin production can use mammalian cells in tissue culture, which are rapidly killed by shiga toxin. Although sensitive and very specific, this method is slow and expensive.

Typically diagnosis has been done by culturing on sorbitol-MacConkey medium and then using typing antiserum. However, current latex assays and some typing antiserum have shown cross reactions with non-E. coli O157 colonies. Furthermore, not all E. coli O157 strains associated with HUS are nonsorbitol fermentors.

The Council of State and Territorial Epidemiologists recommend that clinical laboratories screen at least all bloody stools for this pathogen. The American Gastroenterological Association Foundation (AGAF) recommended in July 1994 that all stool specimens should be routinely tested for E. coli O157:H7.15 It is recommended that the clinician check with their state health department or the Centers for Disease Control and Prevention to determine which specimens should be tested and whether the results are reportable.

Other methods for detecting E. coli O157 in stool include ELISA tests, colony immunoblots, direct immunofluorescence microscopy of filters, as well as immunocapture techniques using magnetic beads. These assays are designed as screening tool to allow rapid testing for the presence of E. coli O157 without prior culturing of the stool specimen.


Antibiotic therapy and resistance

Bacterial infections are usually treated with antibiotics. However, the antibiotic sensitivities of different strains of E. coli vary widely. As Gram-negative organisms, E. coli are resistant to many antibiotics that are effective against Gram-positive organisms. Antibiotics which may be used to treat E. coli infection include amoxicillin as well as other semi-synthetic penicillins, many cephalosporins, carbapenems, aztreonam, trimethoprim-sulfamethoxazole, ciprofloxacin, nitrofurantoin and the aminoglycosides.

Antibiotic resistance is a growing problem. Some of this is due to overuse of antibiotics in humans, but some of it is probably due to the use of antibiotics as growth promoters in food of animals. A study published in the journal Science in August 2007 found that the rate of adaptative mutations in E. coli is "on the order of 10–5 per genome per generation, which is 1,000 times as high as previous estimates," a finding which may have significance for the study and management of bacterial antibiotic resistance.

Antibiotic-resistant E. coli may also pass on the genes responsible for antibiotic resistance to other species of bacteria, such as Staphylococcus aureus. E. coli often carry multidrug resistant plasmids and under stress readily transfer those plasmids to other species. Indeed, E. coli is a frequent member of biofilms, where many species of bacteria exist in close proximity to each other. This mixing of species allows E. coli strains that are piliated to accept and transfer plasmids from and to other bacteria. Thus E. coli and the other enterobacteria are important reservoirs of transferable antibiotic resistance.

Beta-lactamase strains

Resistance to beta-lactam antibiotics has become a particular problem in recent decades, as strains of bacteria that produce extended-spectrum beta-lactamases have become more common. These beta-lactamase enzymes make many, if not all, of the penicillins and cephalosporins ineffective as therapy. Extended-spectrum beta-lactamase–producing E. coli are highly resistant to an array of antibiotics and infections by these strains is difficult to treat. In many instances, only two oral antibiotics and a very limited group of intravenous antibiotics remain effective.

Increased concern about the prevalence of this form of "superbug" in the United Kingdom has led to calls for further monitoring and a UK-wide strategy to deal with infections and the deaths. Susceptibility testing should guide treatment in all infections in which the organism can be isolated for culture.



Phage therapy

Phage therapy—viruses that specifically target pathogenic bacteria—has been developed over the last 80 years, primarily in the former Soviet Union, where it was used to prevent diarrhea caused by E. coli. Presently, phage therapy for humans is available only at the Phage Therapy Center in the Republic of Georgia and in Poland. However, on January 2 2007, the United States FDA gave Omnilytics approval to apply its E. coli O157:H7 killing phage in a mist, spray or wash on live animals that will be slaughtered for human consumption.


Vaccination

Researchers have actively been working to develop safe, effective vaccines to lower the worldwide incidence of E. coli infection. In March 2006, a vaccine eliciting an immune response against the E. coli O157:H7 O-specific polysaccharide conjugated to recombinant exotoxin A of Pseudomonas aeruginosa (O157-rEPA) was reported to be safe in children two to five years old. Previous work had already indicated that it was safe for adults. A phase III clinical trial to verify the large-scale efficacy of the treatment is planned.

In 2006 Fort Dodge Animal Health (Wyeth) introduced an effective live attenuated vaccine to control airsacculitis and peritonitis in chickens. The vaccine is a genetically modified avirulent vaccine that has demonstrated protection against O78 and untypeable strains.

In January 2007 the Canadian bio-pharmaceutical company Bioniche announced it has developed a cattle vaccine which reduces the number of O157:H7 shed in manure by a factor of 1000, to about 1000 pathogenic bacteria per gram of manure.


Role in biotechnology

Because of its long history of laboratory culture and ease of manipulation, E. coli also plays an important role in modern biological engineering and industrial microbiology. The work of Stanley Norman Cohen and Herbert Boyer in E. coli, using plasmids and restriction enzymes to create recombinant DNA, became a foundation of biotechnology.

Considered a very versatile host for the production of heterologous proteins, researchers can introduce genes into the microbes using plasmids, allowing for the mass production of proteins in industrial fermentation processes. Genetic systems have also been developed which allow the production of recombinant proteins using E. coli. One of the first useful applications of recombinant DNA technology was the manipulation of E. coli to produce human insulin. Modified E. coli have been used in vaccine development, bioremediation, and production of immobilised enzymes. E. coli cannot, however, be used to produce some of the more large, complex proteins which contain multiple disulfide bonds and, in particular, unpaired thiols, or proteins that also require post-translational modification for activity.


Model organism

E. coli is frequently used as a model organism in microbiology studies. Cultivated strains (e.g. E. coli K12) are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine. Many lab strains lose their ability to form biofilms. These features protect wild type strains from antibodies and other chemical attacks, but require a large expenditure of energy and material resources.

In 1946, Joshua Lederberg and Edward Tatum first described the phenomenon known as bacterial conjugation using E. coli as a model bacterium, and it remains the primary model to study conjugation. E. coli was an integral part of the first experiments to understand phage genetics, and early researchers, such as Seymour Benzer, used E. coli and phage T4 to understand the topography of gene structure. Prior to Benzer's research, it was not known whether the gene was a linear structure, or if it had a branching pattern.

Long-term evolution experiments using E. coli have allowed direct observation of major evolutionary shifts in the laboratory.

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