Escherichiae Coli: Classification, Structure, Virulence factor, Pathogenesis and Laboratory Diagnosis

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General Characteristics and the History of Discovery


  • A typical member of Enterobacteriaceae family Escherichia coli is a widespread normal inhabitant of human intestinal tract.
  • Escherichiae are permanently discharged into the environment from the gut of mammals, birds, amphibians and many other organisms.
  • These bacteria can be isolated from water, soil and different foodstuffs, including dairy products. Escherichia coli and other coliform bacteria are defined as sanitary indicator microorganisms for these objects.
  • The German scientist T. Escherich discovered E. coli in 1885.

Classification


  • The family Enterobacteriaceae comprises about 50 genera. Among them Escherichia, Shigella, Salmonella, Yersinia, Klebsiella, Proteus, Morganella, Providencia are regarded as the most important in clinical practice.
  • Escherichia genus encompasses at least 6 closely related species. Besides E. coli, Escherichia vulneris can be uncommonly found in wound infections and E. hermannii is rarely isolated from wounds, blood or cerebrospinal fluid (CSF).
  • Escherichia coli species consists of several biotypes and great number of serotypes, which are discerned by their biochemical and antigenic properties.

Structure and Properties of Escherichia coli


Morphology

  • Escherichia coli are small or middle-size gram-negative rod-shaped bacteria 1-3 μm in length. During microscopy they are usually observed as single cells.
  • E. coli carry peritrichous flagella; nevertheless, non-motile microbial forms can be found. They possess numerous pili that promote microbial adherence, nutrition and gene exchange. Escherichiae as well as other enterobacteria have no spores, but can produce capsule.

Cultivation

  • All escherichiae can grow easily on basic nutrient media within standard temperature range 10-45°C at pH of 7.2-7.6.
  • The growth on meat peptone agar (MPA) renders round convex smooth semi-transparent colonies. Meat peptone broth (MPB) cultivation results in diffuse turbidity followed by cell precipitation.
  • As E. coli in most cases utilize lactose, they form lactose-positive colonies, which are red on McConkey agar, Endo agar, and dark-blue on eosin-methylene blue (EMB) agar (i.e., Levine medium).
  • Some E. coli strains produce hemolysin.

Biochemical properties

  • E. coli are facultative anaerobes with mixed type of metabolism. They ferment great number of carbohydrates (glucose, lactose, maltose, mannitol, galactose, and very rarely sucrose) with acid and gas end products.
  • They metabolize proteins with indole formation, express lysine decarboxylase and reduce nitrates to nitrites.
  • As all other members of the family Enterobacteriaceae, E. coli are oxidase-negative, but catalase-positive microorganisms.

Antigenic structure

  • E. coli carries a large variety of antigenic determinants of different origin.
  • Somatic lipopolysaccharide cell wall antigen, or O-antigen, is heat-resistant and withstand heating up to 80-100°С. It demonstrates endotoxin activity due to lipid A moiety, while antigenic epitopes contain predominantly various carbohydrate residues and aminosugars.
  • O-antigen is regarded as group specific. Flagellar H-antigen of E. coli contains protein flagellin, which is heat-sensitive and can be destroyed at a temperature above 56°С.
  • Capsular, or K-antigen is composed of complex polysaccharides. It covers cell wall O-antigen and preserves it against the actions of phagocytes and antibodies.
  • K-antigen of capsular E. coli strains displays evident structural variations: heat-stable A-fraction and heat-labile L- and B-fractions.
  • The complete antigenic structure of E. coli is very individual. Different O-, H-and K-antigens appear in various combinations in particular bacterial strains.
  • More than 170 serogroups based on O-antigen, about 100 types of K-antigen and more than 50 types of H-antigen are known to date.
  • Complete antigenic formula includes the certain O-, H-and K-antigens of the questioned strain (e.g., O26: K60(B6): H2). Antigenic structure of E. coli can be modified by genetic recombinations and mutations that affect the bacteria.

Virulence factors

  • Most of escherichia strains are non-pathogenic, being the representatives of normal human intestinal flora. Some pathogenic isolates (virotypes) possess virulence factors, encoded predominantly by plasmids or temperate bacteriophages.
  • The bacteria produce the number of enterotoxins, hemolysins and cytotoxins (e.g., verotoxins or Shiga-like toxins SLT I and SLT II). Certain virotypes express the structures of type III secretion system (T3SS) or injectisome responsible for microbial invasiveness, spread and intracellular persistence. All microbial cells have the vast number of adhesion molecules.
  • Cell wall LPS exhibits endotoxin activity. Many of E. coli strains produce bacteriocins (colicins), thereby affecting neighboring microflora.

Resistance

  • E. coli stays viable for several months in different environmental conditions. These bacteria are inactivated at temperature of 60°С within 15 minutes and rapidly destroyed by boiling. They are susceptible to most of hospital disinfectants and antiseptics taken in standard concentrations.

Pathogenesis and Clinical Findings of Infections Caused by E. coli


  • The diseases, caused by various escherichia isolates, are generally termed “escherichioses”.
  • Non-pathogenic and facultatively pathogenic E. coli ensure normal intestine functions, taking part in nutrition, cellulose digestion, vitamin synthesis, peristaltic regulation, etc.
  • Nevertheless, they can provoke pathology after colonization of unusual biotopes, such as urogenital or biliary tract, peritoneal cavity or central nervous system. In case ofpatient’s immunodeficiency the generalization of infectious process is possible, resulting in septicemia.
  • Non-specific or opportunistic E. coli infections affect predominantly urinary tract. E. coli is the main causative agent of urinary infections, especially in young women. These bacteria are shown to produce hemolysins.
  • E. coli, expressing pili of certain type (P pili), become associated with pyelonephritis, since P pili promote microbial adhesion to epithelium of urinary tract.
  • Bacterial adherence to uroepithelium is followed by microbial degradation with the release of LPS endotoxin. It leads to neutrophil infiltration, cytokine overproduction and progression of inflammatory response.
  • E. coli strains are often isolated in patients with cholecystitis, cholangitis, appendicitis, peritonitis and other abdominal diseases.
  • Also E. coli is an important cause of infant meningitis. It mainly affects premature newborns and infants under the age of 1 month.
  • Most of causative agents pertain to the specific serovar O18:K1 as K1 capsular antigen is resistant to complement action. In addition, these bacteria have special S-fimbriae with elevated tropism to endothelial cells of CNS blood vessels. The disease is extremely severe; the mortality rate may exceed 50%.
  • Specific E. coli infections comprise a large number of diarrheal diseases. They are caused by pathogenic escherichia strains.
  • These disorders have specific mechanisms of development resulting from the action of various E. coli toxins.
  • Infection are transmitted by fecal-oral route (foodborne, waterborne or through contaminated fomites).
  • The main sources of infection are sick persons or carriers. In situations with enterohemorrhagic diarrheas there can be carrier animals (e.g., cattle).
  • Enteropathogenic E. coli (EPEC) affect infants worldwide. EPEC coli-enteritis is caused by numerous serotypes of E. coli. EPEC reveal a distinct capacity of adherence to intestinal cells.
  • Intestinal colonization by EPEC is promoted by interaction of microbial adhesin intimin with its specific receptor Tir. This process is mediated by bacterial type III secretion system (injectisome) that initially translocates an intimin receptor Tir into the host cell (see below).
  • As the result, membrane cup-like filamentous pedestals enwrapping each bacterium are formed that is followed by destruction of local microvilli. This is known as an attaching and effacing (A/E) phenomenon.
  • All of the genes essential for induction of A/E lesions in EPEC are confined within specific “pathogenicity island” termed as the locus for enterocyte effacement.
  • Devastation of epithelial villi results in profound watery diarrhea.
  • Enterotoxigenic E. coli (ETEC) is the common causative agent of diarrhea in developing countries. Also it causes so-called “traveller’s diarrhea”. The infectious dose here is relatively high – about 106-109 microbial cells.
  • ETEC infection results from the action of heat labile exotoxin (enterotoxin) with molecular weight of 80,000 that is very similar with cholerogen of Vibrio cholerae.
  • B-subunit of enterotoxin is absorbed to the intestinal cells via cell membrane ganglioside receptor. Subunit A enters into the cell across the membrane promoting ADP-ribosylation of cellular G-proteins.
  • This event activates guanylate cyclase and adenylate cyclase resulting in abnormal increase of cAMP concentration inside the cells. The latter event stimulates secretion of chlorides into the small intestine with impairment of sodium and water absorption. As the result, massive non-inflammatory diarrhea evolves.
  • Certain isolates express several heat stable enterotoxins. Co-expression of both toxin types results in more profound diarrhea.
  • Enterotoxins, as well as colonization factors of ETEC, are encoded predominantly by plasmid genes.
  • Heat labile exotoxin elicits the synthesis of antitoxic antibodies, which possess neutralizing activity.
  • Enteroinvasive E. coli (EIEC) reveal the striking ability to invade intestinal epitheliocytes with intracellular propagation that is followed by microbial lateral spread towards adjacent neighboring cells.
  • The enteroinvasive disorders are very similar to shigellosis caused by Shigella flexneri, S. boydii and S. sonnei. EIEC are almost identical to shigella (non-motile bacteria, which lack of lactose fermentation) but they are deprived of ability to produce Shiga STX toxins.
  • Enteroaggregative E. coli (EAEC) promote diarrhea due to their strong adhesive capacity to intestinal cells with tendency of self-aggregation. The mode of their adhesion is not similar with adherence pattern of EPEC.
  • EAEC binding was primarily determined as diffuse adherence, but further two main patterns were observed: aggregative adherence and diffuse adherence. Bacteria express two types of specific fimbriae: aggregative adherence fimbriae I and II (AAF/I and AAF/II, respectively).
  • Usually EAEC don’t produce toxins. Nevertheless, they damage intestinal cells and hamper the normal exchange of water and electrolytes within the bowel, thus causing a chronic or persistent form of diarrhea with duration of more than 14 days.
  • Enterohemorrhagic E. coli (EHEC) are the most dangerous representatives of coliform bacteria. They cause severe hemorrhagic colitis with diarrhea and intestinal cell destruction.
  • The infection outcome becomes much more serious with the development of life-threatening hemolytic uremic syndrome (HUS) that is often followed by acute renal failure.
  • The incubation period of the disease endures about 5-7 days.
  • The infectious dose of bacteria is extremely low (1-100 cells).
  • For a long time E. coli of O157:H7 serotype was considered to be the major pathogenic variant of EHEC. Now it is obvious that STEC strains causing human disease, pertain to a very broad range of O:H serotypes (more than 30 O-serogroups with multiple antigenic variants are known to date, and this list is being increased permanently).
  • High virulence of EHEC depends on production of adherence factors and potent cytotoxins.
  • The ability of EHEC as well as EPEC to attach to and efface enterocytes results mainly from the activity of E. coli outer membrane protein ”intimin” and its translocated receptor Tir.
  • Both proteins are encoded by genes located in the same “locus of enterocyte effacement” within pathogenicity island of bacterial chromosome.
  • Before tight bacterial attachment, Tir receptor protein is injected into intestinal cells via type III bacterial secretion system (needle complex, or injectisome). Once expressed on enterocyte membranes, Tir interacts with microbial intimin that ensures the strong binding of E. coli to intestinal cells.
  • EHEC secrete two main types of Shiga-like toxins (SLT I and SLT II or verotoxins), which are very similar with Shigella dysentheria exotoxin Stx (SLT I toxin is almost identical). Toxin production in EHEC is encoded by temperate bacteriophages.
  • SLT toxin of EHEC is composed of A and B subunits. Protein A-subunit of 32 kDa is noncovalently bound to five 7-kDa B-subunits. B-subunit promotes attachment to eukaryotic cell receptor glycolipid Gb3 (or globotriosyl ceramide).
  • The cells, bearing Gb3 receptor, are susceptible to SLT toxin action. When Shiga-like toxins appear in the bloodstream, they induce the damage of glomerular endothelial cells of kidneys, which express large amounts of the Gb3 receptor.
  • After receptor binding, toxin molecules are internalized by receptor-mediated endocytosis. Subunit A is further cleaved into two fragments, where A1 portion of toxin renders RNA N-glycosidase activity.
  • A1 fragment breaks down N-glycosidic bond in the 28S rRNA, thus preventing aminoacyl-tRNA binding to the 60S subunit of ribosome. These molecular events terminate elongation of protein sequence and eventually cause cell death.
  • Additional virulence factors encompass several enterohemolysins and extracellular serine protease, which cleaves human coagulation factor V, thereby maintaining hemorrhagic colitis.
  • Multiple devastating activities of EHEC virulence factors promote destructive hemocolitis with severe diarrhea.
  • The disease tends to be self-limited, though hemolytic uremic syndrome, followed by hemolytic anemia and thrombocytopenia, can develop in 10-30% of cases and even more. It is one of the leading causes of renal failure in children. The mortality rate in HUS is near 5%.
  • Immunity reactions after escherichia infections are usually group specific and low grade. Natural passive immunity conferred by maternal milk sIgA can protect newborns and infants against coli-enteritis for several months after birth. Similarly, trans-placental IgG-mediated immunity defends infants against infections, caused by some enteroinvasive escherichia strains.
  • Normal microflora of gastrointestinal tract (e.g. bifidobacteria and lactobacilli) promotes powerful non-specific host defense due to their substantial antagonistic activity against pathogenic enterobacteria.

Laboratory Diagnosis of Coli-Enteritis and Other E. coli Infections


  • Specimens for diagnosis of non-specific escherichioses are obtained from the site of infection: urine, bile, blood, pus, or wound discharge are examined.
  • For laboratory diagnosis of escherichia-associated diarrhea feces, vomiting masses, food remnants, water, washing samples, etc. should be examined.
  • For rapid identification of pathogenic E. coli in clinical specimens molecular genetic tests are applied (e.g., PCR test).
  • Microscopical tests are useless owing to the evident morphological similarities of all enterobacteria.
  • To confirm the diagnosis of coli-enteritis, isolation of microbial culture is elaborated.
  • To aim this, the specimens are plated upon the differential or selective nutrient media (McConkey agar, EMB or Endo medium). The growth of colored lactose-positive colonies is evaluated.
  • To determine the nature of grown E. coli isolates, the tentative slide agglutination test with polyspecific OK-serum against the most widespread enteropathogenic E. coli is performed.
  • At least 10 lactose-positive colonies should be investigated. In case of positive results the rest of the colony is planted on slant MPA to obtain pure culture. It is identified by slide agglutination tests with different serovar-specific OK-sera.
  • Positive result of slide agglutination with type-specific OK-serum is confirmed by extended tube agglutination test. To establish the concordance of isolated culture to serum specifity and titer, the reaction is performed separately for O- and K-antigens (with boiled and native culture, respectively).
  • Evaluation of biochemical properties, phage typing and antibiotic susceptibility tests accomplish culture examination.
  • To reveal enterohemorrhagic E. coli, the specimens are planted on modified McConkey agar that contains sorbitol instead of lactose. EHEC 0157:H7 are sorbitol-negative, whereas other escherichia are usually sorbitol-positive on MacConkey agar.
  • Shiga-like toxins as well as escherichia enterotoxins are determined by ELISA, cell culture tests or molecular genetic methods in reference laboratories.

Treatment and Prophylaxis of E. coli Infections


  • Various groups of antibiotics (amoxycillin, third-generation cephalosporins, aminoglycosides, or fluoroquinolones) are used for treatment of opportunistic infections, caused by escherichiae.
  • In patients with E. coli-associated diarrheas an adequate antibiotic therapy shortens the diarrheal period, but the microbial resistance rapidly increases under antibiotic pressure.
  • Antibacterial treatment should be administered with great precautions in patients with hemorrhagic colitis and HUS. The drugs affecting metabolism of microbial nucleic acids (e.g., fluoroquinolones and co-trimoxazole) are not recommended here as they may stimulate the spreadof virulence genes among the enterobacteria. In these clinical situations carbapenems are regarded as the most suitable antimicrobial agents.
  • The disease therapy with probiotics (e.g., lactobacterin, bificol, bifidumbacterin) is also beneficial.
  • Specific prophylaxis is not available for E. coli infections. The tight control of sanitary conditions, prevention of water and foodstuff contamination, maintaining of hygiene standards is of great importance.