Enterobacteriaceae are a family of Gram-negative bacteria that includes many common pathogens found in the human gut and environment. They are rod-shaped, often motile, and test negative for oxidase but positive for catalase. Many species ferment glucose and reduce nitrates. There are over 100 species currently classified in the family. Escherichia coli is a prominent member and common gut bacterium, but also causes urinary tract infections, diarrhea, and other infectious diseases through various virulence factors.

1. ENTEROBACTERIACEAE
Dr V S Vatkar
Associate Professor
Microbiology Department
D Y Patil Medical College, Kolhapur

2. ∗ Found in soil & water.
∗ Some of them are predominant
bacterial flora of human intestine &
animals.

3. ∗ Gram negative, non sporing, motile or
non motile bacilli, capsulated or non
capsulated
∗ Grows aerobically or anaerobically.
∗ Oxidase negative.
∗ Catalase positive.
∗ Ferment glucose & other carbohydrates:
produce acid or acid & gas.
∗ Reduces nitrates to nitrites.
DEFINITION

4. ∗ In 1972 Edward & Ewing described 11
genera & 26 species.
∗ In 1985 Farmer & associates described
22 genera & 69 species & 29 enteric
groups.
∗ Now 31 genera & 139
species,bigroups,unnamed enteric
groups.
TAXONOMY

5. Lactose fermenters: E.coli,Klebsiella
Late lactose fermenters:
Shigella sonei,paracolons
Non lactose fermenters: Salmonella &
Shigella
All LF enteric bacilli : known as COLIFORM BACILLI,
majority of intestinal commensals are LF(colon bacilli
& E.coli).
OLD CLASSIFICATION
Lactose fermentation on MacConkey Agar

6. EDWARDS & EWING CLASSIFICATION
∗ TRIBE I : ESCHERICHIAE
Genus :
• Escherichia
• Shigella
• TRIBE III :
• SALMONELLAE
• Genus :
• Salmonella
∗ TRIBE II :
EDWARDSIELLAE
Genus:
• Edwardsiella
• TRIBE IV :
• CITRIBACTERIACEAE
• Genus :
• Citrobacter

7. Classification ( contd )
∗ TRIBE V
KLEBSIELLEAE :
Genus:
Klebsiella,
Enterobacter, Serratia,
Hafnia & Pantoea
TRIBE VII
YERSINIEAE :
Genus
Yersinia
∗ TRIBE VI :
PROTEAE :
Genus:
•Proteus, Providencia,
Morganella
TRIBE VIII
ERVINEAE :
Genus
Ervinia

8. N.A. Low,convex,smooth/muciod,grey colonies.
B.A. Same as N.A. some show hemolysis,
swarming(Proteus)
∗ M.A. LF or NLF colonies
Special Media like:
- DCA LF or NLF colonies( salmonella & Shigella)
- Eosin Methylene Blue Agar(EMB): Green sheen
( E.coli)
CHARACTERISTICS
FOR PRESUMPTIVE IDENTIFICATION

9. ∗Mainly by Biochemical reactions
•Glucose fermentation
•Cytochrome oxidase NEGATIVE
•Nitrates : reduced to Nitrites
•IMViC tests
•Motility test
SCREENING CHARACTERISTICS

10. CARBOHYDRATE UTILISATION
∗Glucose
fermentation
∗Lactose
fermentation
mainly
ACIDS: Lactic acid,
Acetic acid, carbonic
acid or Formic acid
GAS : Hydrogen, CO2,
N2.

11. OXYDASE TEST
∗Enterobacteriac
eae bacilli give
test
NEGATIVE(E.coli)
∗ Reagents :
tetramethyl-para-
phenyl diamine or
dimethyl para
phenyl diamine
hydrochloride.

12. NITRATE REDUCTION TEST
∗ All Enterobacteriaceae
gr.give test +ve
except : Enterobacter
aggtomerans,some
Serratia spp. Yersinia.
∗ Indicators:
∗ ά-nephthalamine &
sulfanilic acid.
∗ RED color +ve E.coli
∗ Yellow color – ve
Acinetobacter

13. PHENYLALANINE DEAMINASE
PRODUCTION TEST
∗Differentiation of
proteus from
other GNB.
∗Indicator : Ferric
chloride
∗GREEN color +ve
test

14. TSI & H2S TEST
∗L F: Yellow slant &
butt.
∗NLF: Red slant &
Yellow butt.
∗Non fermenters:
Red slant & butt.
∗H2S: Yellow butt,
Red slant, black
color on slant.

15. INDOLE TEST
∗Kovac’s reagent
or Ehrlich’
reagent used as a
indicator.
∗Red color ring +ve
test

16. METHYL RED TEST
∗Ferment glucose
to form pyruvate
∗Indicator : Methyl
red
∗Red color +ve test

17. V-P TEST
∗ Ferment glucose to
form Glycol
∗ Indicators : alc soln
of ά-nephthol &
KOH
∗ Red / Pink color +
ve test

18. CITREATE TEST
∗ Simmon’s citrate
agar
∗ Indicator :
Bromothymol blue
∗ Blue color + ve test

19. UREASE TEST
∗Christensen
medium / Stuart’s
urea broth
∗Indicator : Phenol
red
∗Purple pink color
+ve

20. ∗ Help to identify Late Lactose Fermenting
organisms.( colorless +ve,yellow – ve)
∗ E.coli & Shigella sonei, Salmonella spp.&
Citrobacter spp. ( E.coli +ve & proteus –
ve,control)
DECARBOXYLATION OF
LYSINE,ORNITHIN,ARGININE :
Variable
ONPG & β GALACTOCIDASE TEST

21. Escherichia coli
∗ Named after the
scientist ESCHERICH
(1885)
∗ Present in human &
animal GIT.
∗ Detection of E.coli in
drinking water shows
recent pollution of water
with human or animal
faeces.
∗ E fergusonii, E hermanii, E
vulneris: little medical
importance

22. MORPHOLOGY
∗ GNB , STRAIGHT
RODS
∗ SIZE 1-3×0.4-0.7µm
∗ MOTILE :
peritrichous flagella
∗ Non-sporing
∗ Capsule & fimbria in
some strains.

23. Gram stain

24. ∗ AEROBES & FACULTATIVE ANAEROBES
∗ TEMP 10 – 40ºC , optimum temp 37ºC
∗ Colony characters : large, whitish, smooth,
opaque, emulsify in saline ( S ). Rough
colonies : irregular, dull, autoagglutinable in
saline ( R ). S – R variation : repeated
subcultures, loss of virulence factor( surface
Ag ).
∗ Broth : general turbidity.
∗ DCA & SS media : Inhibition of growth
CULTURAL CHARACTERISTICS

25. Colonies on M.A. & CLED

26. Colonies on EMB medium

27. XLD MEDIUM
( Xylose Lysine Deoxycholate agar )
∗ Yellow colonies of
E.coli.

28. BIOCHEMICAL REACTIONS
∗ Ferments
Glucose,Lactose,Ma
nnitol, Maltose
∗ Indol: +ve, MR: +ve,
VP: – ve
∗ Citrate : – ve,
∗ H2S: – ve
∗ urease: – ve
∗ Gelatin : not
liquefied
∗ ONPG test : + ve
∗ Oxidase : - ve
∗ Nitrate reduction :
+ ve
∗ Phenyl Alanine
Deaminase : - ve
∗ KCN medium: no
growth

29. * O’ Ag (somatic) : 170 Ag recorded, inhibit phagocytosis
∗F Ag : fimbrial Ag, heat labile, detached when
heated to 100 o C
∗‘K’ Ag (capsular): 100 , acidic polysaccharide, located
in the ENVELOPE/ Micro capsule. 3 parts : L Ag, A Ag, B Ag.
Later shown that B Ag is not a separate entity.
∗‘H’ Ag (flagellar) : 75
* Normal intestinal strains : K Ag absent, has Early ‘O’ gr.
(1,2,3,4 etc)
*Pathogenic strains : Later ‘O’ gr (26,55,86,111 etc)
ANTIGENIC STRUCTURE

30. ∗ Surface Ag :
a) Somatic Lipo-polysaccharides O Ag :
- resist PHAGOCYTOSIS
- Endotoxic activity
- Prevent bactericidal effect of complement
b) Envelope or K Ag :
- resist PHAGOCYTOSIS
- Prevent antibacterial effect in serum
VIRULENCE FACTORS ( TWO TYPES)

31. c) Fimbriae :
- Chromosomally determined : non
pathogenic
- Plasmid coded : small in no. & acts as
virulence factor
- OR colonization factor Ag (CFA) : in
enterotoxogenic E.coli causes Diarrhoea.
- In UTI: P fimbria binds specifically to P
bl.gr.substance on human RBCs &
uroepithelial cells.
SURFACE Ag (cond)

32. ∗EXOTOXINS:
- Hemolysin: produce by most of the virulent
strains than avirulent strains.
- Enterotoxin: a) Heat Labile Toxin (LT):
complex polysaccharide subunits : 1 A
subunit: active
5 B subunits : binding. Produced by ETEC
b) Heat Stable Toxin : STa(STI) & STb(STII) ETEC
c) Verotoxin (VT) also called Shiga like toxin
,produced by EHEC.
TOXINS

33. Toxin binds to Gm1 receptors of intestinal epithelium
with subunit B subunit A activated
activates Adenyl
cAMP cyclase.
increase outflow of
water & electrolytes leads to DIARRHOEA
in intestinal lumen
*
MOA of Cholera toxin is same as LT
Mode Of Action of LT

34. ST I binds to cGMP activation of
cGMP
in intestine.
fluid accumulation in
intestine.
Mode Of Action of ST

35. ∗ Cytotoxic effect on Vero cells , HeLa
cells
∗ Similar to Shigella dysenteriae type 1
toxin so called Shiga like toxin
∗ VT genes : phage coded
∗ A & B subunits
∗ Antigenically different VT is called VT2,
not neutralized by Shiga like toxin VT1.
Verotoxin (VT)

36. ∗Urinary Tract Infection (UTI)
∗DIARRHOEA
∗PYOGENIC INFECTIONS
∗SEPTICEMIA
CLINICAL INFECTIONS

37. ∗Acquired after Diagnostic
instrumentation, prostate
enlargement, renal calculi,
pregnancy ( Asymptomatic
bacteriuria)
∗Serotypes : ‘O’ gr 1,2,4,6,7.
Urinary Tract Infection

38. ∗ Asymptomatic bacteriuria : asymptomatic inf
during pregnancy may cause PYELONEPHRITIS later
in pregnancy, HT leads to prematurity & perinatal
death of fetus.
∗ Lower UTI mainly due to fecal contamination &
may not have K Ag ( organism causing cystitis).
Isolates from pyelonephritis carry K Ag
(ascending inf)
∗ DIAGNOSIS OF UTI :
- Normal urine is sterile, but during voiding it may
contaminate by genital commensals.
UTI

39. ∗ Sample collection
- Collect urine by catheterizations to avoid
contamination (sometimes lower UTI may ascend
to cause ascending inf.) .
- CLEANE VOIDED MIDSTREAM sample of urine is
collected.
- Transport to lab without any delay. Delay more
than 1-2 hrs REFRIGERATE the sample.
- Quantitative culture : most of the specimen
contain 10,000 to 1,00,000 bact / ml. Active inf may
contain 1,00,000 bact / ml. this level is SIGNIFICANT
BACTERIURIA. Patient on antibiotics, diuretic drugs,
bact like staph , low count may be significant.

40. ∗ Griess Nitrate test :
absence of nitrate in normal urine. Presence
of nitrates: nitrate reducing bact.
∗ Catalase test :
effervescence on adding H2O2. +ve result
seen in hematuria
∗ Triphenyl tetrazolium chloride(TTC) test : detects
respiratory activity of growing bacteria.
Pink ppt
∗ Microscopy: GNB seen
∗ Glucose test paper :
utilization of minute amt of glucose present
in urine by bact.
SCREENING TESTS for UTI for presumptive
diagnosis

41. ∗Deep slide culture method: agar
coated glass slide is immerged in urine
sample , incubate, growth is estimated by
colony counter or by color change of
indicator.
(none of the screening test is as sensitive /
reliable as culture).
CULTURE : Two methods
- Pour Plate method
- semiquantitative culture method : standard
loop method

42. ∗ Isolates are identified by biochemical
reactions
∗AST : development of drug resistance
frequently with E.coli inf.

43. ∗ First isolated from infant suffering from enteritis.
∗ Bary 1945: found the role E.coli (sp.type) during
hospital out breaks of childhood diarrhoea in London.
∗ TYPES OF DIARRHOGENIC E.coli :
a) Enteropathogenic E.coli (EPEC)
b) Enterotoxogenic E.coli (ETEC)
c) Enteroinvasive E.coli (EIEC)
d) Enterohaemorrhagic E.coli (EHEC)
e) Enteroaggregative E.coli (EAEC)
DIARRHOEA

44. Pathogenesis of EPEC
∗ Bacteria attaches to
the mucosa of S.I.
∗ Then attaches to the
cup like projections
(pedestals) of host cell
membrane.
∗ Distruption of brush
border (microvilli)

45. Pathogenesis of ETEC

46. Pathogenesis of E.coli

47. ETEC O sero
gr
6,8,15,25,27
,63,78,115,
148,153,159
Infant &
adult
watery
diarrhoea
traveler's
diarrhoea
Bact adhere
to intestinal
mucosa by
CFA(colonis
ation factor
Ag),
releases
LT / ST or
both
Typing with
sp antisera,
demo of
toxin in
tissue by LA
ELISA,
genetic
probes for
ST & LT
EIEC 28ac,112ac
,
124,136,143
,144,152,16
4
Dysente
ry like
disease
all ages
Epith cell
invasion
Typing with
sp antisera.
definative:He
La/HEp-2 cell
inv assay.
Virulence
Marker
Ag(plasmid
coded)
detected by
ELISA.
Sereny test

48. VCEC
Food
poisoning
(canned
food)
O 157:H 7
O 26: H 1
Bloody
diarrhoea,
HC,HUS
VT1 & VT 2
or Shiga like
toxin, affects
vascular
endothelium,
renal lesion
& capillary
microangiop
athy.
Typing with
sp
antiserum,VT
1 & 2 probes,
demo of VT 1
& 2 in Vero
cells(cytotoxi
c effect)
EPEC O gr.
18,26,44,55,
86,111,114,
119,125,126
,127,128,14
2
Diarrhoea
in infants,
rarely in
adults
Pathogen
esis
Unknown
Typing with
sp
antisera,EAF
probes,
demo of
adhesion in
HeLa,HEp-2
cell

49. ∗ Persistent diarrhoea, in developing countries
∗ Produces heat stable enterotoxin 1(EAST 1)
∗ O : untypeble H : typable
∗ Aggregate in ‘Stacked Brick’ form on HEp-2 cells
∗Diffusely Adherent E coli: less well
established as pathogen
EAEC

50. ∗Intra abdominal infections :
Peritonitis, abscesses from
spillage of bowel contents.
∗Perianal inf
∗Neonatal meningitis
∗Wound inf
PYOGENIG INFECTIONS

51. ∗SEPTICEMIA :
Blood stream
invasion causes
septic
shock(endotoxic
shock), systemic
inflammatory
response
syndrome (SIDS)
∗Antibiotic
sensitivity test :
- organism shows
resistance to
multiple drugs like
Penicillin,
Cephalosporins,
Quinolone gr etc,
- AST is most imp for
the treatment.

52. Detection of O157: H 7
∗ Detection of Shiga like toxin
directly from stool.
∗ Direct plating on SMAC
(Sorbitol MacConkey Agar).
OR
∗ Cefixim SMAC OR
∗ Cefixim & Tellurite SMAC OR
media containing 5 bromo-5
chloro-3 indoxyl-β-D
glucuronide OR 4
methylumbelliferyl-β-D
glucuronide.
∗ Immunogenic separation by
using O 157 sp Ab coated
beads after culture.

53. ∗ E.fergusonii : isolated from stool
∗ E.hermanii : isolated from wd, sputum,
stool. Agglutinate with E.coli O 157
antiserum
∗ E.vulneris
∗ E.blatte
Other Escherichia spp