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E.coli Dr. Mahadi
- 1. Faculty of Medical Laboratory Sciences Department of Microbiology Medical Microbiology course Dr. Mahadi Hassan Mamoud PhD Medical Microbiology
- 2. Enterobacteriaceae • Commonly present in large intestine. • Nonsporing , Non Acid fast,Gram – bacilli. • A complex familyof organisms. • Some are non pathogenic. • A few are highly Pathogenic. • Some commensals turn out to be pathogenic.as in UTI after catheterization.
- 3. Characters of Enterobacteriaceae • All Enterobacteriaceae • Gram-negative rods • Ferment glucose with acid production • Reduce nitrates into nitrites • Oxidase negative • Facultative anaerobic • Motile except Shigella and Klebsiella • Non-capsulated except Klebsiella • Non-fastidious • Grow on bile containing media (MacConkey agar)
- 4. Enterobacteriaceae: Genetic Properties • Chromosomal DNA has 39-59% guanine-plus- cytosine (G+C) content • Escherichia coli is the type genus and species of the Enterobacteriaceae • Species of Enterobacteriaceae more closely related by evolutionary distance to Escherichia coli than to organisms of other families (Pseudomonadaceae, Aeromonadaceae)
- 5. Identification of Enterobacteriaceae Biochemical reactions • Oxidase test • All members of Enterobacteriaceae are oxidase negative • Pseudomonas is oxidase positive • O/F test • All members of Enterobacteriaceae are O+/F+ • Pseudomonas is O+/F- • Nitrate reductase • All members of Enterobacteriaceae are nitrate reductase positive • Pseudomonas is nitrate reductase negative
- 6. Classification of Enterobacteriaceae Enterobacteriaceae Lactose fermenters E. coli, Citrobacter, Klebsiella, Enterobacter Non-lactose fermenter Salmonell, Shigella Proteus, Yersinia There are several selective and differential media used to isolate distinguishes between LF & LNF The most important media are: MacConkey agar Eosin Methylene Blue (EMB) agar Salmonella Shigella (SS) agar In addition to Triple Sugar Iron (TSI) agar
- 7. Differentiation between LF & NLF by Growth on MacConkey agar MacConkey agar is selective & differential medium for Enterobacteriaceae MacConkey Agar Contains Bile salts Crystal violet Lactose Neutral red Inhibit growth of G+ve bacteria Cause of selectivity Cause of differential pH indicator Acidic: Pink Lactose feremnters Pink colonies Lactose non feremnters colorless colonies
- 8. Classification of Enterobacteriaceae according to lactose fermentation (growth on MacConkey Agar) Enterobacteriaceae Lactose Fermenters Lactose Non-Fermenters Escherichia coli Klebsiella spp Enterobacter spp Citrobacter spp Salmonella spp Schigella spp Proteus spp Yersinina spp Pink colonies Colorless colonies Acid Neutral red No acid
- 9. Escherichia coli • Named by Escherichia • Wide group of bacteria on basis of Bio typing and Serotyping Produce infections in Humans and Animals Detection of E.coli in water indicates pollution and contamination.
- 10. Classification • Domain: Bacteria • Kingdom: Bacteria • Phylum: Proteobacteria • Class: Gamma Proteobacteria • Order:Enterobacteriales • Family: Enterobacteriaceae • Genus: Escherichia • Species: Escherichia coli (E.coli)
- 11. E.coli • Morphology Gram - ve Straight rods, • 1-3 X 0.4 -0.7 microns, • Appear in singles or in pairs, • Motile by peritrichate flagella. • Very few strains non motile • Not spore forming, Non acid fast.
- 12. Cultural characters • Aerobic / Facultative Anaerobic • Grows between 10 – 40 c optimal at 37 c • Grown in simple medium • Produce Large grayish ,Thick white , moist smooth opaque colonies • May contain capsule. • On MacConkey medium Produce Bright pink Lactose fermenters.
- 13. • O antigen – Somatic (on LPS) – 171 antigens • H antigen – Flagella – 56 antigens • K antigen – Capsule and or fimbrial antigen – 80 antigens O18ac:H7:K1 18th O antigen 1st K antigen 7th H antigen Antigenic structure
- 14. E.coli Biochemical Characters IMViCtests. Indole +ve MethylRed +ve Voges Proskauer – ve Citrate –ve Urease –ve KIA yellowbutt yellowslant Acid&Gas NoH2S Set ofSugar Glucose A&G Lactose A&G Mannitol A&G Maltose A&G Motility Motile
- 15. Pathogenesis • Adhesion of the microorganisms to the intestinal epithelial cells • Production of enterotoxins Heat labile (LT) and Heat stable (ST) • Verotoxin production of cytopathic effects in green monkey kideny cells • Invasion of epithelial cells
- 16. • Heat labile toxin • like choleragen • Adenyl cyclase activated • cyclic AMP • secretion water/ions • Heat stable toxin • Guanylate cyclase activated • cyclic GMP • uptake water/ions
- 17. Urinary tract infection Is the leading cause of urinary tract infections which can lead to acute cystitis (bladder infection) and pyelonephritis (kidney infection). E. coli is the most common cause of urinary tract infection. Community- vs. hospital-acquired UT infection Symptoms: urinary frequency, dysuria, hematuria, and pyuria. Can result in bacteremia and sepsis. clinical diseases
- 18. Other infection with E.coli • Neonatal Meningitis. particularly K1 strains • Pyogenic infections. • Intraabdominal infections • Peritonitis. Abscess. • Septicemias • Produce Drug resistant infections.
- 19. • Certain strains of E. coli • ingested in sufficient quantities by host. • enteritis, enterocolitis, and colitis • pathogenic strains of E. coli which cause disease in the intestine are obligate pathogens
- 20. six distinct "pathotypes" of E. coli (which cause disease in intestine) • Enteropathogenic E. coli (EPEC). • Enterotoxigenic E. coli (ETEC). • Shiga toxin-producing E. coli (STEC)/enterohemorrhagicE. coli (EHEC), Verocytotoxin-producing E. coli (VTEC). • Enteroinvasive E. coli (EIEC). • Enteroaggregative E. coli (EAEC) (EAggEC) • diffuselyadherentE. coli (DAE)
- 21. Enteropathogenic E. coli • EPEC is main cause of infant diarrhea • Breast-feeding diminishes the incidence of EPEC infection. • Rapid person-to-person spread may occur. • After infection with EPEC there is loss of microvilli. • Symptoms : Fever, Diarrhea (NO Blood), Vomiting, Nausea. • Confirm with Polyvalent and monovalent sera.
- 22. Enterotoxigenic E. coli • ETEC is a major cause of endemic diarrhea in children during the first 3 years of life. • ETEC is the most common agent of traveler's diarrhea. • There is production enterotoxin • Produce Heat stable /Heat labile toxins. • Present with Nausea, Vomiting and Lose stool
- 23. Enterotoxigenic E.coli •Laboratory Diagnosis Demonstration of Enterotoxin LT and ST Tissue culture tests. ELISA. Passive agglutination tests. Animal experiments inRabbit ileal looptest. Con.
- 24. Enteroinvasive E. coli EIEC a relativelyuncommoncauseof diarrhea. Some are non motilestrains. EIEC sharesmany genetic andclinical featureswith Shigella. The diseaseresembles the shigellosis The difference EIEC produces disease only at a large inoculum(108 to 1010 CFU8), with onset generally occurringafter an incubationperiod of 1 to 3 days.
- 25. Enteroinvasive E. coli EIEC Sereny test: • Instillationof a suspension of freshly isolated EIEC or Shigella into theeyes of guineapigs leads to mucopurulent conjunctivitis and severe keratitis. • Mice can also be used. CellPenetrationin HeLa or HEP-2 cells. Plasmid detection: VMA ELISA: The plasmid codes for outer membraneantigens called the virulence markerantigens(VMA) which can be detected by the ELISA (VMA ELISA) test. Con.
- 26. Enterohemorrhagic E.coli Produce Verocytotoxin or shiga like toxin • Named so because it was first detected by its cytotoxic effecton Vero cells, a cellline derived from African green monkeykidney cells. • It is also known as SLT = shiga liketoxin because it is similar to the shigella dysenteriae type 1 toxin in its physical, antigenic and biological properties Mild diarrhea - can be fatalhemorrhagic colitis. and uremic syndrome. Presentin Human and Animal feces. Hemorrhagic complication withO157 in Japan and USA.
- 27. Lab. Diagnosis o Culture o DNA detection methods o Cytotoxic effects on Vero cells. o Detection with monovalent sera O157/H7 Enterohemorrhagic E.coli Con.
- 28. Dr.T.V.Rao MD 28
- 29. • Haemolytic anaemia • Acute renal failure • Thrombocytopenia The verotoxin enters the blood stream EnterohemorrhagicE.colican cause Haemolytic Uremic Syndrome HUS Enterohemorrhagic E.coli
- 30. Enteroaggregative and Diffusely Adherent E. Coli EAEC and DAEC primarily in developing countries and in young children. These strains can cause traveler's diarrhea. A largeinoculum is required for infection. In vitro, the organisms exhibit a diffuse or "stacked-brick" adherence pattern. Can cause Diarrhea Detect by Culture methods Brick-like aggregates on cell surfaces Mucus biofilm inhibits fluid absorption Diarrhea Detection of Enterotoxin
- 31. Culturing for E.coli • Mid stream sample/semiquantitativeculturing (Kasset al) >_ 1.00,000/ml of urine. ( significantBacteriuria) • Urine shouldnot be kept in wardsfor > 2 hours andto be preserved at 4 c • Culture by standardloopmethod. • Fixedvolume cultured onMacConkeyagar Lactose fermenters I M Vi C • Antibioticsensitivitytested.
- 32. Escherichiacolias a Genetic tool. • The study of Escherichia coli and its plasmids and bacteriophages has provided a vast body of genetical information, much of it relevant to the whole of biology. This was true even before the development of the new techniques, for cloning and analysing DNA, that have revolutionized biological research during the past decade.. Much of the background of knowledge necessary for the cloning and expression of genetically engineered information, as well as the techniques themselves, came fromwork with thisorganism.
- 33. Why E.coliis preferred • E. colicells only have about4,400 genes whereas the human genome project has determined that humans containapproximately30,000 genes. • Also, bacteria,includingE. coli, live their entire lifetimein a haploidstate, with nosecondalleleto mask the effects of mutationsduring proteinengineering experiments.
- 34. specimens • Urine • Stool. • Blood • CSF Lab Diagnosis
- 35. • Microscopy • make distinction is between inflammatory and noninflammatory disease • Culture • Identification ofserotypes/pathotypes Usuallyperformed atreference lab TheE.coliisolate treated ,standardized andallowedto react with antisera (knownantibodies) in antigenantibodyreactions anddesignated Specialtests to detect LT and ST are notavailablein mostclinicallaboratories Serological characters: Somatic(O) antigen. Flagellar (H) antigen. Manystrainshave capsular(K) antigen.
- 36. Lab Diagnosis • Grossly bloody or mucoidstool suggests aninflammatory process. • A test for fecal leukocytes (preparation of a thin smear of stool on a glass slide, addition of a drop of methyleneblue,and examinationof the wet mount)can suggestinflammatory disease in patientspresentingwithdiarrhea, • A testfor fecal lactoferrin, whichis a marker of fecal leukocytes, is more sensitive and is available in latex agglutinationand enzyme-linkedimmunosorbentassay formats.
- 38. 1/24/2020 (Gram stained film of E. coli)
- 39. 1/24/2020 (E. coli on MacConkey’s agar)
- 40. 1/24/2020 (Lactose fermenters and non lactose fermenters on MacConkey’s agar)
- 41. Reasons E. coli is used for Gene Cloning • The microorganism Escherichia coli has a long history of use in the biotechnology industry and is still the microorganism of choice for most gene cloning experiments. Although E. coli is known to the general population for the infectious nature of one particular strain (0157:H7) few people are aware of how versatile and useful E. coli is to genetic research. There are several reasons E. coli became so widely used and is still a common host for recombinant DNA.
- 42. Genetic Simplicity • Bacteria make useful tools for genetic research because of their relatively small genome size compared to eukaryotes. E. coli cells only have about 4,400 genes whereas the human genome project has determined that humans contain approximately 30,000 genes. Also, bacteria, including E. coli, live their entire lifetime in a haploid state, with no second allele to mask the effects of mutations during protein engineering experiments.
- 43. Growth Rate • Bacteria typically grow much faster than more complex organisms. E. coli grows rapidly at a rate of one generation per twenty minutes under typical growth conditions. This allows for preparation of log-phase (mid-way to maximum density) cultures overnight and genetic experimental results in mere hours instead of several days, months or years. Faster growth also means better production rates when cultures are used in scaled up fermentation processes.
- 44. Safety • E. coli is naturally found in the intestinal tracts of humans and animals where it helps provide nutrients (vitamins K and B12) to its host. There are many different strains of E. coli that may produce toxins or cause varying levels of infection if ingested or allowed to invade other parts of the body. Despite the bad reputation of one particularly toxic strain (O157:H7), E. coli are generally relatively inocuous if handled with reasonable hygiene.
- 45. Conjugation and the Genome Sequence • The E. coli genome was the first to be completely sequenced. Genetic mapping in E. coli was made possible by the discovery of conjugation. E. coli is the most highly studied microorganism and an advanced knowledge of its protein expression mechanisms makes it simpler to use for experiments where expression of foreign proteins and selection of recombinants is essential.
- 46. Ability to Host Foreign DNA • Most gene cloning techniques were developed using this bacterium and are still more successful or effective in E. coli than in other microorganisms. E. coli is readily transformed with plasmids and other vectors, easily undergoes transduction, and preparation of competent cells (cells that will take up foreign DNA) is not complicated. Transformations with other microorganisms are often less successful.