1. E coli, Klebsiella and Enterobacter
Prof M.I.N. Matee
School of Medicine
MUCHS

2. Escherichia coli Normal inhabitant of the G.I. tract
Some strains cause various forms of gastroenteritis.
Is a major cause of urinary tract infection and neonatal meningitis and septicemia.

3. Morphology and culture characteristics
Gram negative rods
Non-spore former, non-caspulated and motile
Culture characteristics
N.agar – circular, convex, small colonies
MacConkey medium– rose pink
Eosin Methylene blue – Metallic sheen colonies

4. Biochemical reactions
IMViC: ++--
TSI: Acid butt/acid slant with gas production

5. Antigenic structure Has O, H, and K antigens.
K1 has a strong association with virulence, particularly meningitis in neonates.

6. Antigenic structure.
Complex
Lipopolisaccharides/Somatic or O antigen
Heat stable.more than 150 types
Most external in the cell wall
detected by bacterial agglutination
Antibody produced is predominantly IgM

7. Capsular/K antigen Flagella /H antigen
Sometimes external to O antigen but not always
Can be polysaccharides or protein
Flagella /H antigen
Heat and alcohol labile.

8. Colicines/Bacteriocines
Produced by many Gram -ves
Virus like bactericidal substance
Active against some other bacteria of similar
or closely related species

9. E. coli toxins Virulence factors Toxins
Enterotoxins – produced by enterotoxigenic strains of E. coli (ETEC). Causes a movement of water and ions from the tissues to the bowel resulting in watery diarrhea. There are two types of enterotoxin:
LT – is heat labile and binds to specific Gm1 gangliosides on the epithelial cells of the small intestine where it stimulates production of cAMP.
Increased cAMP alters the activity of sodium and chloride transporters producing an ion imbalance that results in fluid transport into the bowel.

10. E. coli toxins
ST – is heat stable and binds to specific receptors to stimulate the production of cGMP with the same results as with LT.

11. Urinary tract infections
E coli is the most common cause of UTI
Account for 90% of cases in young women
Symptoms
Frequency, dysuria, hematuria, pyuria
UTI may lead to bacteremia and sepsis
Nephropathogenic E coli produces hemolysin

12. E coli associated diarrhoeal diseases

13. Enterotoxigenic E. coli
Heat labile toxin
like choleragen
Adenyl cyclase activated
cyclic AMP
secretion water/ions
Heat stable toxin
Guanylate cyclase activated
cyclic GMP
uptake water/ions

14. A.Enterotoxigenic E coli ( ETEC)
Common cause for travelers diarrhoea, and watery diarrhoea in children.
Colonisation factor facilitates the attachment to the intestinal epithelium.
Some ETEC produces heat labile exotoxin LT and heat stable or either of the toxins

15. B.Enteropathogenic E coli (EPEC)
Important cause of diarrhoea in infants of
developing countries.
Adhere to mucosal cells in small bowel,loss of microvilli, NONINVASIVE
enter to cell body. result in watery diarrhoea.
Self limiting ,can be chronic.
Normally do not produce toxins

16. C. Enterohemorrhagic E coli ( EHEC)
Produce verotoxin which has similarities to Shiga toxin
Associated with hemorrhagic colitis, severe form of diarrhoea.
Hemolytic uremic syndrome Disease can be prevented by thorough cooking.

17. Escherichia coli O157:H7
This strain produces a powerful toxin, verotoxin, which can cause severe illness and death.
E coli O157:H7 does not ferment sorbitol and is negative on sorbitol MacConkey agar
Other Shiga toxin producing serotypes (e.g. O111 and O26) are also in the family of enterohemorrhagic E. coli and can cause similar disease.
Hemorrhagic colitis – preventable by thorough cooking of meat

18. Hemorrhagic colitis(HC) or bloody diarrhea and abdominal cramps
Lasts for 5-10 days usually
Infection may proceed to Hemolytic Uremic Syndrome (HUS), a severe cytopathic attack on the kidneys requiring intensive care and dialysis.
Red blood cells are destroyed requiring blood tranfusion

19. D.Enteroinvasive E coli (EIEC)
Produces disease similar to shigellosis.
In adults this has been isolated with Shigella
Commonly affect children in developing countries,
and travelers.
Disease is due to invasion into mucosal cells of the intestine
multiply inside the cells and destruction /inflammation/ulceration
diarrhoea with blood
EIEC are nonlactose fermenter,or late lactose fermenter
and non motile.

20. E. Enteroaggregative E coli (EAEC)
Produce acute/chronic diarrhoea in persons in developing countries.
Sepsis When normal host defense is poor, sepsis can happen. Common in new born babies whose IgM level is low.

21. Treatment of E.coli related diarrhoea
1st Line
Nitrofurantoin
Nalidixic acid
Norfloxacin
Ampicillin
Cotrimoxazole
2nd line
Ciprofloxacin/Ceftriaxone/Cefuroxime
Gentamicin

22. Meningitis
E coli and Gp.B Strept. are the leading causes for
meningitis in infants.
K1 antigen is responsible for meningitis
K1 cross reacts with the Gp.B capsular
polysaccharides of N meningitides.

23. Pneumonia 25% of gram -ve pneumonia with 50% mortality
Usually broncho pneumonia
High level of resistance to Ampicillin /Cotrimoxazole

24. Klebsiella spp

25. Morphology and culture characteristics
Gram negative rods
Non-spore former, caspulated and non-motile
Culture characteristics
N.agar – mucoid, circular, convex, small colonies
MacConkey – mucoid, rose pink

26. Biochemical reactions
IMViC: --++
TSI: Acid butt/acid slant with gas production

27. K pneumoniae and K oxytoca Hospital acquired infections due to
K rhinoscleromatis produces rhinoscleroma,
condition with destructive granuloma of the nose and
pharynx.
K ozenae – progressive atrophy of nasal mucous membrane

28. Klebsiella
K pneumoniae
Present in respiratory tract and feces of about 5% of
normal individuals.
Can cause bacterial pneumonia (3%).
Produce extensive hemorrhagic necrotising consolidation of lungs.

29. Enterobacter aerogenus Capsulated Free living in the intestine
Cause UTI and sepsis.
Motile
Citrate +
Ornithine decarboxylase
Produce gas from glucose
Ferments lactose
VP + (like Klebsiella)

30. Laboratory identification tests

31. Diagnosis of Bacterial Infection
microscopy
unstained or stained with e.g. Gram stain
Stain
Decolorise
Counterstain
culture
identification by biochemical or serological tests on pure growth from single colony
on plates or in broth
sensitivities
by disc diffusion methods, breakpoints or MICs
Serodiagnosis
DNA technologies

32. Gram negative rods

33. Lactose fermentor on MCA

34. Lactose positive Klebsiella sp

35. E. coli

36. EMB is both selective and differential

37. Indole production
Degradation of tryptophan by tryptophanase to produce indole
Red colour develops after adding a solution containing p-dimethylaminobenzaldehyde (Kovacs or Erlichs)
Negative
Positive

38. Triple Sugar Iron Agar (TSI):
Purpose: To differentiate bacteria based on their ability to ferment glucose, lactose and/or sucrose, and to reduce sulfur to hydrogen sulfide.

39. INTERPRETATION OF TUBES ABOVE
TUBE 1 (UNINOCULATED)
TUBE 2
TUBE 3
TUBE 4
TUBE 5
SLANT - A K
BUTT
HYDROGEN SULFIDE +
GAS
A=Acidic K=Alkaline

40. ONPG test Determines presence of -galactosidase
Utilizes o-nitrophenyl-d--galactosidase
Differentiates late lactose fermenting organisms
Positive Negative

41. Methyl Red
This is a qualitative test of the acidity produced by the growth of a bacterium in phosphate –buffered glucose peptone water
E coli produces a pH of about 5, and hence a red colour is produced after addition of methyl red.
Enterobacter aerogenes the pH never drops so low, it appears yellow after addition of methyl red.

42. Voges-Proskauer
This test for production of acetylmethylcarbinol (AMC) from carbohydrates by bacteria in glucose phosphate peptone water
Red colour is produced after addition of alpha naphthol and KOH
Klebsiella +
E coli -

43. MR/VP continued Reading Results: VP: left + and right –
MR— a + result is red (indicating pH below 6) and a – result is yellow (indicating no acid production)
VP—A + result is red after VP reagents are added (indicating the presence of acetoin) and a – result is no color change.
VP: left + and right –
Methyl Red: left – and right +

44. IMViC test
IMViC test is a group of four biochemical tests collectively used for primary identification of enteric bacteria
Indole
Methyl red
Voges proskuer
Citrate utilization test

45. Indole (IMViC tests) • E. coli (pink/red) + • E. coli (left side) –
• Kovac’s reagent
detects if tryptophan
has been hydrolyzed
to indol/tryptophanase

46. Methyl Red (MR) (IMViC tests)
Enterobacter
aerogenes (left) –
E. coli (bright red) +
Reagent: Methyl red
indicator identifies pH
change due to mixed
acid fermentation

47. Voges – Proskauer (VP) (IMViC tests)
Enterobacter aerogenes +(left)
E. coli – (right)
Red colour is produced after addition of alpha naphthol and KOH
Klebsiella +
E coli -

48. Citrate utilization
Ability to use sodium citrate as sole source of carbon
Medium has sodium citrate, ammonium salt, bromothymol blue
Positive test - deep blue colour in 24-48hrs
Indicates citrate utilization
Production of alkaline products
E. coli (left green) –
• Enterobacter aerogenes
(right royal blue) +
Positive Negative

49. Treatment of enteric bacteria
No specific treatment is available
A number of antibiotics could be used
Marked antibiotic resistance due to plasmids
May involve surgical correction
Treatment of shock
Environmental sanitation

52. Thank you

53. References: