E coli

Microbiology of E coli giving basic of Escherichia coli, its morphology, cultural and biochemical characteristics, Antigenic character, pathogenesis, laboratory diagnosis, prevention and control

1. Anup Muni Bajracharya (Escherichia coli)

2. Introduction Gram negative, facultative anaerobic, coliform, lactose fermenter, gas producer bacteria belonging to the family enterobacteriaceae . Normal flora of intestine so called as coliforms A re beneficial to us helps in producing Vitamin K2 , p revents colonization of other bacteria It causes a broad spectrum of disease in humans such as enteric illness, UTI, neonatal sepsis, neonatal meningitis, hemolytic uremic syndrome(HUS). Acts as the indicator for the fecal contamination of water.

3. Morphology Shape: rod shaped, bacillus. Size: 1-3 × 0.4-0.7µm in size. Colony characteristics: On N utrient agar- it produces large, circular, low convex, grayish white, moist, opaque and smooth colonies. On MacConkey agar- it produces bright pink colonies due to the lactose fermentation. On Blood agar- it produces beta hemolytic colonies. It is motile by peritrichous flagellae , though some strains are non-motile. Spores are not formed. Capsules and fimbriae are found in some strains.

4. Culture: G rows at temperature range of 10-40º(optimum 37º) and pH 7.2. Grows on a wide range of media including Muller-Hinton agar, Nutrient agar, Blood agar and MacConkey agar. Primary isolation can be made on Nutrient agar and Blood agar. On N utrient agar, it produces large, circular, low convex, grayish white, moist, opaque and smooth colonies. On MacConkey medium, it produces bright pink colonies due to the lactose fermentation. On Blood agar, it produces beta hemolytic colonies . In liquid broth culture, it produces turbid growth.

5. Biochemical tests: Indole Methyl red Voges-proskaur Citrate + ve + ve - ve - ve

6. Cell wall components: Cell wall is made up of heat stable lipopolysaccharide(LPS), cytoplasmic membrane, thin layer of peptidoglycan. The lipopolysaccharide consists of three components, the genus specific somatic O polysaccharide, a core polysaccharide commonly present in all and and lipid A.

7. Antigenic structure: It possess four major antigens. H or flagellar antigens : It is heat and alcohol liable proteins present on flagella. Usually it is genus specific antigens and not shared by all. O or somatic antigens : It occurs on the surface of the outer membranes and are determined by specific sugar sequences on the cell wall. It is an LPS complex and integral part of the cell wall. Heat stable, resistance to boiling up to 2hrs 30min. It also shows cross reactions with related genera. K or capsular antigens : It is the acidic polysaccharide antigen present in the envelope or micro capsule of bacteria. It encloses O antigens and makes virulence by inhibiting phagocytosis.

8. d) F or fimbrial antigens : It is present on the fimbriae and are heat-liable proteins. A number of filamentous protein structures resembling fimbriae have been described in E.coli . K88 , K99 antigens of E.coli stains are enterotoxigenic and causes diarrhea in human and animals. This fimbrial antigens also contributes virulence factor in bacteria.

9. Pathogenesis: E.coli is invasive bacterium. It colonizes the human intestine and under specific condition directly invades the intestinal mucosa or produces toxins to cause intestinal infections. The bacteria can enter the blood stream and cause septicemia, meningitis and other systemic manifestation. The bacteria under certain conditions, directly invade urinary tract causing UTIs or cause intra abdominal infections.

10. Virulence factors: E.coli produces several virulence factors which includes the following: Common virulence factor associated with Enterobacteriaceae . These includes the various factors such as fimbriae, endotoxin , capsule, and growth factors. 2. Specialized virulence factors associated with E.coli : Diseases Causative organism Traveler’s diarrhea Enterotoxigenic Escherichia coli (ETEC) Infant diarrhea Enteropathogenic Escherichia coli (EPEC) Enteroaggregative Escherichia coli (EAEC) Diffuse aggregative Escherichia coli (DAEC) Hemorrhagic colitis(HC) Enterohemorrhagic Escherichia coli (EHEC) Dysentery Enteroinvasive Escherichia coli (EIEC) Haemolytic uremic syndrome (HUS) Enterohemorrhagic Escherichia coli (EHEC

11. Enteroinvasive E.coli : EIEC strains closely resembles S higella in many ways. EIEC strains are non motile. They do not ferment lactose or ferment late with production of acid only. They do not decarboxylate lysine decarboxylase. They have capacity to invade the interstitial epithelial cells and also penetrate HeLa cells in tissue culture. The ability to invade the cells by EIEC is determined by a large plasmid, which codes for outer membrane antigens called virulence marker antigen (VMA). The bacteria lyse the phagocytes and multiply in the cell cytoplasm which leads to the epithelial cell destruction along with inflammatory infiltration and develops ulcers in the intestine.

12. Enterohemorrhagic E.coli : EHEC strains are the most common cause of gastrointestinal infections in the developed countries. These strains produces diarrheal disease ranging in severity from mild uncomplicated diarrhea to fatal hemorrhagic colitis. EHEC disease is mostly common in children below 5 years and in summer months. The disease is caused due to the ingestion of contaminated water, unpasteurized milk or fruit juices, uncooked vegetables and fruits contaminated with humans and animal feces. Serotype O157:H7 and O26:H1 are the EHEC strains that causes the disease.

13. These strains produces the shiga toxins (Stx-1 and Stx-2), which are responsible to cause diarrheal disease. Stx-2 is most commonly associated with hemolytic uremic syndrome(HUS), thrombocytopenia, microangiopathic hemolytic anaemia . Sxt-2 also causes the destruction of glomerular endothelial cells, resulting in reduced glomerular filtration and acute renal failure. The toxins also stimulate production of tumor necrosis factor- α and interleukin-6 which contributes further to the disease process.

14. Enteroaggregative E.coli : EAEC are also so called because they show a typical ‘stacked brick’ arrangement on Hep-2 cells or glass due to their autoagglutination . These EAEC strains secrete a low molecular weight, heat stable enterotoxin-1. They increases the mucus secretion, which forms a layer overlying the epithelium of the small intestine. And this layer of biofilm traps bacteria in epithelium of the small intestine. In animal they causes shortening of the microvilii , mononuclear infiltration, and haemorrhage . These strains are associated with persistent, watery diarrhea with dehydration in the infants in developing countries.

15. Diffusely adherent E.coli : DAEC strains causes watery diarrhea found primarily in children below 1 and 5 years of age. These strains are identified by their ability to adhere to cultured cells. They cause the elongation of the microvilli with the bacteria trapped in the cell membrane.

16. Clinical syndromes: Urinary tract infections: E.coli is most common bacterium responsible for causing more than 80% of all community acquired UTI. E.coli cause a wide range of UTIs including uncomplicated urethritis, symptomatic cystitis, pyelonephritis, acute prostatitis, prostatic abscess or urosepsis . Uncomplicated cystitis occurs primarily in sexually active females who are colonized by uro -pathogenic strains of E.coli . The peri -urethral region is colonized by E.coli due to the fecal contamination, and bacteria reach to the urinary bladder during sexual intercourse.

17. Gastroenteritis: EPEC, EAEC and DAEC causes watery diarrhea and dysentery. EPEC primarily affects infants and children by causing acute watery diarrhea, which causes dehydration or become chronic and lead to the failure to thrive. ETEC is widely distributed to the areas with poor sanitation and is a ubiquitous contaminants of food and water sources. It is the primary cause of traveler’s diarrhea and major cause of infantile diarrhea in underdeveloped countries. EIEC produce diarrhea and dysentery, disease similar to that caused by Shigella species. These strains cause watery diarrhea, dysentery, fever, vomiting, painful abdominal cramps and stool often contains blood and leukocytes.

18. EHEC is an important cause of food borne illness, particularly in developed countries such as US and Canada. EHEC causes two distinctive syndromes hemorrhagic colitis and hemolytic uremic syndromes (HUS) by producing two Shiga toxins. Hemoragic colitis: E.coli O157:H7 is the most common strain to cause EHEC diarrhea which has a small infective dose (around 100 bacilli) and spreads easily from child to child by the fecal oral route. Hemolytic uremic syndrome: it is a disease caused by abnormal destruction of RBCs and thrombocytopenia. And it is a very serious life threatening complication seen in the 10-15% of children suffering from EHEC diarrhea. The condition typically arises in 2 nd weeks of illness after the child has recovered from diarrhea.

19. Neonatal meningitis: E.coli are an important cause of meningitis in neonates. In adults, it occurs following neurosurgical procedures, CNS trauma, or complicating strongyloides stercoralis hyperinfection involving CNS. Neonatal meningitis has a high mortality rate of 8%. Other infestions : These includes intra abdominal infections due to E.coli , often resulting from a perforated appendix, diverticulum, or due to intra-abdominal abscess, endophthalmitis , sinusitis, endocarditis, or skin and soft tissue infections are the other pyogenic infections caused by E.coli .

20. Epidemiology: Many strains of e.coli primarily affects populations in developing countries. There is wide variation in prevalence of the strains world wide. ETEC are the primary cause and major cause of travelers diarrhea and are the major cause of infantile diarrhea in poor and developing countries. ETEC causes nearly more than 600million cases of diarrhea every year and 700,000 deaths in children below 5 years. EHEC are an emerging cause of food borne disease particularly in the northern United states and Canada. Outbreaks of HUS in children that caused fatalities have also been documented in these countries.

21. Reservoir, source, and transmission of infection: Most of the E.coli infections exception of neonatal meningitis and gastroenteritis are endogenous. The E.coli that causes infection are patients normal flora which some times causes UTI due to the poor personal hygiene or when patients defense are compromised. Diarrhea caused by E.coli is wide spread in the areas with poor sanitations and infections are exogenous. Food contaminated with human or animal feces causes the diarrheogenic E.coli . The usual source of E.coli in neonatal infections is the intestinal tract of mother .

22. Laboratory diagnosis: Isolation of E.coli by culture. Demonstration of toxins of diarrheogenic E.coli . Specimens: Urine is the specimen of choice for diagnosis of UTI caused by uropathogenic E.coli . Mid-stream urine should be taken for culture. Other specimen include feces or rectal swab for gastroenteritis, blood for septicemia, cerebrospinal fluid(CSF) for meningitis, sputum for pneumonia and other body fluids such as pus from wounds.

23. Culture: Definitive diagnosis is based on the isolation of E.coli from various clinical specimen by culture. Urine culture is very useful procedure for diagnosis of UTI. Stool culture is widely used to isolate diarrheogenic E.coli . Blood, CSF, and other body fluids culture is carried out depending upon clinical disease caused by E.coli . Urine culture: It is the semiquantitative method for the culture of E.coli and other gram negative bacteria from urine sample and is widely used method in microbiology laboratory in case of UTI. In this method mid-stream urine is taken in a sterile container and is immediately transfer to the laboratory.

24. A predetermined quantity of urine is inoculated into the MacConkey and blood agar with the standarized innoculating loop of 0.05ml. After the over night incubation at 37ºC pink, flat colonies in MacConkey agar and beta-hemolytic colony in blood agar are obtained. If the no. of colonies in MacConkey agar after over night incubation is 500 then the viable bacterial count per ml of urine is (500×200) 100,000 or 10 5 and if the bacteria are more than this limit then the patient is suffered from UTI.

25. Other specimens for culture: CSF culture positive for E.coli establishes the diagnosis of E.coli meningitis. Isolation of the organism from blood, pus and other specimens is definitive for diagnosis of infections caused by E.coli . After the culture of the various clinical specimens, different strains of E.coli can be identified by serotypes, animal inoculation, cytopathic effects in cell culture or molecular methods. Identification of EPEC: Specific serogroups of E.coli (O26, O55, O86, O111, O114, O125, O126, O128 and O142) are most commonly associated with outbreaks of EPEC. Therefore E.coli colonies isolated from feces on MacConkey agar are identified by agglutination test with specific polyvalent and monovalent antisera.

26. Identification of ETEC: Diagnosis of ETEC diarrhea depends upon the demonstration of enterotoxin in E.coli isolates from stool because toxin production is not associated with specific serogroups of E.coli The strain of ETEC may produce either liable or stable toxins or both. Many tests are available for the demonstration of liable or stable toxins produced by ETEC and therefore are used for the detection and identification of E.coli isolates from stool as ETEC. Identification of EIEC: Many of the EIEC strains are typical E.coli strain. They are non motile and do not ferment lactose or ferment it late with production of acid but not gas. They also do not decarboxylate lysine.

27. EIEC are further identified by ; Sereny test: it is carried out in guinea pigs by installation of isolated EIEC into the conjunctival sac of guinea pigs and is examined after 72 hours for mucopurulent conjunctivitis and severe keratitis. Cell culture test: it is carried out in the HeLa or HEP-2 cells. The bacterial suspension is added to the mono-layers of cells. The cells are then examined for the presence of intracellular E.coli because if EIEC is present then it penetrates the cells and replicates inside it. VMA-enzyme linked immunosorbent assay: this is the serological test used to detect the plasmid, which codes for outer membrane antigens called VMA, in stool isolates of EIEC.

28. Identification of EHEC: E.coli O157:H7 is the most common serotype associated with the clinical disease caused by EHEC strains. The strain typically does not ferment sorbitol. Hence sorbitol MacConkey medium is frequently used for isolation of the strain from stool by culture. Identifications of other strains: EAEC strains are identified by agglutination tests with specific antisera . Most of them are not typed by O antisera, but by specific H antisera.

29. Treatments: E.coli isolated from community acquired infections are usually sensitive to commonly used antibiotics except penicillins . Hospital acquired E.coli isolates, however, show multidrug resistance. UTI and neonatal sepsis are treated with appropriate antibiotic therapy. Treatment of bacterial gastroenteritics is primarily supportive. Third generation of cephalosporins such as ceftriaxone are recommended for meningitis and pneumonia caused by E.coli .

30. Prevention and control: Availability of safe drinking water, proper food hygiene and sanitary disposal of excreta are the most cost-effective strategies for reducing the incidence of enteric infections caused by E.coli . Cooking ground beef thoroughly is the most effective way to prevent hemorrhagic colitis caused by E.coli O157:H7.

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