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  1. Enterobacteriaceae Escherichia coli

  2. Escherichia coli • Ferment lactose • Indole positive • Most strains flagellated • Some strains have capsules • Some strains  haemolytic on sheep blood agar • Identified/confirmed to species using API 20E

  3. E. coli 24 h, aerobic Sheep blood agar MacConkey

  4. Laboratory Diagnosis • Must only be isolated from carefully taken samples; e.g. cervical swabs, mastatic milk, midstream urine. • Isolation: culture on both Blood and MacConkey agars. • Pathogenic strains are often haemolytic on blood agar. • Strong lactose fermenters Pink colonies on MacConkey agar. • On EMB, show unique characteristic green metallic sheen. • Antibiotic sensitivity test. • For enterotoxogenic strains, demonstration of enterotoxins, using ELISA. • Biochemical tests: API 20E, or IMViC tests (indole/methyl red/ VP/ citrate). E-coli is (+/+/-/-)

  5. API 20E

  6. Laboratory Diagnosis IMViC is reliable for E-coli, as no other lactose +ve Enterobacteriaceae give such a combination of results

  7. E. coli and disease • Neonatal and post weaning diarrhoea – many species • Neonatal septicaemia – many species • Mastitis – many species • UTI – many species • Haemorrhagic colitis (HC) + Haemolytic uraemic syndrome (HUS)- man • Watery mouth - lambs • Infection of existing lesions- many species

  8. E.coli: the versatile pathogen • Enterotoxigenic E.coli ETEC –animals, humans • Enteropathogenic E.coli EPEC,– animals, humans • Verotoxigenic E.coli VTEC,– animals, humans (Enterohaemorraghic E.coli EHEC) • Extraintestinal Pathogenic E. coli ExPEC –animals, humans • Non-enteric infections: Urinary tract infections, mastitis, septicaemia, meningitis

  9. E.coli serotypesand disease

  10. E.coli pathotypes • ETEC • Heat labile enterotoxin (LT), heat stabile enterotoxin (ST), particular fimbriae • EPEC • Pathogenicity island (LEE), type III secretion system, intimin, particular fimbriae • EHEC • As EPEC + Vero toxins (VT) [Shiga-like toxins (SLT)] • ExPEC. Septicaemia, UTI strains • Haemolysin, iron-uptake systems, particular fimbriae and capsules • , Mastitis

  11. E.coli pathotypes - determination Identify virulence factors • Serological • Antibodies against virulence factors • Molecular • DNA probes • PCR

  12. Capsule Siderophore Fimbriae (Pili) Specific types Fe3+ Aerobactin Toxins Haemolysin CNF-1 Virulence factors of extraintestinal isolates of E.coli

  13. Fe Fe Aerobactin Not all E.coli strains produce In animal strains usually on plasmid Fe Fe Siderophores in E.coli Enterochelin (enterobactin) Produced by all E.coli strains Genes on chromosome Affinity for iron in vitro: enterochelin > aerobactin

  14. Enterotoxigenic E.coli (ETEC) • Non-invasive • Non-inflammatory, watery diarrhoea • Young of many animal spp. cattle, sheep, etc • Travellers diarrhoea humans - different strains • Non-zoonotic

  15. Disruption of electrolyte balance Watery diarrhoea Pathogenesis of enterotoxigenic E.coli diarrhoea

  16. Attachment of ETEC to the jejunum of a calf

  17. E.coli fimbriae

  18. E.coli Fimbriae/Pili - ETEC Fimbriae involved in enteric infections • K88 = F4: pigs • K99 = F5: pigs, sheep, calves • 987p= F6: pigs and calves • F41: pigs and calves Human ETEC different fimbriae Other pathotypes – different fimbriae are important

  19. Disturbances in electrolyte transport and as a consequence water absorption Guanylate cyclase [cGMP] [cAMP] ADP-ribose Adenylate cyclase GS Water balance in the gut changes from net absorption to net secretion of water GS + NAD Action of heat labile (LT) and heat stabile (ST) toxins ST LT

  20. Enterotoxins produced by E.coli Heat labile toxin (LT) - Highly immunogenic A subunit = 30kD Enzymatic = ADP-ribosyltransferase B subunit = 55kD. 5 subunits of 11kD. Binds receptors, GM1 ganglioside and lactosyl glycoproteins

  21. Enterotoxins produced by E.coli Heat stable toxin (ST) - Non-immunogenic Single small peptide. 2 types: STa, ~ 2kD, methanol soluble, produced by animal and human E.coli strains STb, ~5kD, methanol insoluble produced by porcine E.coli strains

  22. Diarrhoea: ETEC v Salmonella sp. • Remain extracellular • Non-inflammatory diarrhoea • Invade cells • Inflammatory diarrhoea • E.coli • Salmonella sp.

  23. Enteropathogenic E.coli (EPEC) • Diarrhoea in lambs, calves, piglets, puppies and children • Attaching and Effacing (AE) lesion • Pedestal

  24. Verotoxigenic E.coli (VTEC) • Some E.coli produce toxin that is cytotoxic for Vero cells in vitro = vero toxin (VT). • Homologous toxin 1st identified in Shigella sp. • verotoxin toxin also referred to as Shiga-like toxin (SLT). • Therefore Verotoxigenic E.coli (VTEC) or shigella-like toxin producing E.coli (STEC). Because certain strains cause haemorrhagic colitis in humans also called Enterohaemorrhagic E.coli (EHEC).

  25. Verotoxins/Shiga-like toxins A = Enzymatic, N-glycosidase Inhibits protein synthesis: Kills cells Removes an adenine residue from the 3’ end of 28S rRNA. Prevents binding of aminoacyl t-RNA to the ribosome. B = Binding. Gangliosides Gb3 and Gb4 • Main target –endothelial cells of blood vessels • Oedema • Haemorrhage • Thrombosis

  26. Enterohaemorrhagic E. coli (EHEC) Hemorrhagic Colitis (more than 90% of cases): • Sudden onset of sever cramps and abdominal pain • Bloody diarrhoea • Nausea and vomiting • Little or no fever. Haemolytic uraemic syndrome (HUS) • Most commonly occurs following infection in children • 2-7% of E. coli O157:H7-infected children develop HUS. • Haemolytic anaemia • Thrombocytopenia • Renal failure • Antibiotics may make worse!

  27. EHEC • Typical Symptoms of EHEC Infections • 1. GI tract → Large Intestines. Bacteria travels through GI tract and attaches itself to inside wall of the large intestines. • 2.Binds tightly via the same attachment-effacement mechanism and cup-like pedestals as EPEC.The toxins secreted by EHEC then causes inflammation of the intestinal walls, and is believed to result in hemorrhagic colitis. • 3. Hemorrhagic Colitis: • a)      Sudden onset of sever cramps and abdominal pain • b)      Diarrhea within 24 hrs. • c)      Diarrhea becomes watery. • d)      Diarrhea becomes grossly bloody; bloody to the naked eye. • e)      Nausea and vomiting • f)      Little or no fever.

  28. EHEC E. coli O157:H7 was first isolated in 1982. • Hamburger Disease. • Many other foods. • including raw potatoes, raw milk, unpasteurized fruit juices and apple cider, lettuce/alfalfa sprouts/salads (at least 11 outbreaks in the U.S. since 1995), and possibly even sea eels in Japan! • Reservoir for EHEC: 1.      Mostly in intestine of cattle 2.      Also found in chickens, deer, sheep and pigs. 3.      Does not make the carrier animal sick. 4.      Meat typically becomes contaminated during the slaughtering process. 5. Bacteria on cow’s udders and equipment can also contaminate milk. • EHEC occurs more in developed countries, both outbreaks and sporadic, peaks in summer season.

  29. EHEC pathogenesis Diarrhoea AE lesions VT HC, HUS