Enterobacteriaceae

description

1. Dr.Babasaheb N. Kumbhar
M.V.Sc
[email protected]
ENTEROBACTERIACEAEENTEROBACTERIACEAE

2. EnterobacteriaceaeEnterobacteriaceae
Habitat
digestive tube(colon) of human and animals
150 Species
Facultative anaerobes
Diarrheal illnesses
3 million death/year
4 billion infectoions/ worldwide

3. Enterobacteriaceae
Classification – more than15 different genera
Escherichia
Shigella
Edwardsiella
Salmonella
Citrobacter
Klebsiella
Enterobacter
Hafnia
Serratia

4. Enterobacteriaceae
Proteus
Providencia
Morganella
Yersinia
Erwinia
Pectinobacterium

5. Family Enterobacteriaceae
(nonmotile)
(nonmotile)
True pathogen
True pathogen
True pathogen
Certain E .coli strains
can be considered
true pathogens

6. Morphology and General
Characteristics
Are facultative anaerobes
Gram-negative, non-sporing, rod shaped bacteria
If motile, motility is by peritrichous flagella(Except
Shigella & Klebsiella)
Many are normal inhabitants of the intestinal tract of
man and other animals
Some are enteric pathogens and others are urinary or
respiratory tract pathogens
Differentiation is based on biochemical reactions and
differences in antigenic structure

7. Morphology and Physiology
Short gram-negative rods.
Facultative anaerobes.
Grow readily and rapidly on
simple media.
K. pneumoniae
Klebsiella spp. have large capsule
(form large and very mucoid colonies);
those of Enterobacter have smaller
capsule; the others produce diffusible
slime layers (form circular, convex and
smooth colonies).

8. ENTEROBACTERIACEAE PHYSIOLOGY
Glucose is fermented with strong acid
formation and often gas
Reduce nitrates to nitrite
Do not liquify alginate
Oxidase negative
Basis for speciation within a family
- differences in carbohydrate they ferment
- variations in end-product production
- variation in substrate utilization

9. Enterobacteriaceae
Opportunistic pathogens
Escherichia coli
Klebsiella pneumoniae
Enterobacter aerogenes
Serratia marcescens
Proteus spp.
Providencia spp.
Citrobacter spp.
Obligate pathogens
Salmonella spp.
Shigella spp.
Yersinia spp.
Some E. coli strains
Sepsis
Meningitis
UTI
Diarrhea
Pneumonia

10. Incidence of Enterobacteriaceae
Associated with Bacteremia

11. Sites of Infections with Members of the
Enterobacteriaceae

12. CULTURAL CHARACTERISTICS
On non differential or nonselective media
blood agar or infusion agar
- no species distinction
- appear as moist, smooth, gray colonies
Selective media -To isolate Shigella and
salmonella from fecal matter
Differential media – selectively inhibit
gram-positive organisms and to separate
enterics in broad categories

13. Modes of Infection
•Contaminated food and water (Salmonella spp.,
Shigella spp., Yersinia enterocolitica, Escherichia coli
O157:H7)
•Endogenous (urinary tract infection, primary bacterial
peritonitis, abdominal abscess)
•Abnormal host colonization (nosocomial pneumonia)
•Transfer between debilitated patients
•Insect (flea) vector (unique for Yersinia pestis)

14. Urinary Tract Infection, Pneumonia
•Escherichia coli, Klebsiella pneumoniae, Enterobacter
spp., and Proteus mirabilis
•Pneumonia: Enterobacter spp., Klebsiella pneumoniae,
Escherichia coli, and Proteus mirabilis
•Wound Infection: Escherichia coli, Enterobacter spp.,
Klebsiella pneumoniae, and Proteus mirabilis
•Bacteremia: Escherichia coli, Enterobacter spp.,
Klebsiella pneumoniae, and Proteus mirabilis
Intestinal Infection
•Shigella sonnei (serogroup D)
•Salmonella serotype enteritidis
•Salmonella serotype typhimurium
•Shigella flexneri (serogroup B)
•Escherichia coli O157:H7
•Yersinia enterocolitica

15. MacConkey (MAC) Agar

16. Eosin Methylene Blue (EMB) Agar (Levine)

17. Antigenic StructureAntigenic Structure
–Most are motile by Most are motile by peritrichous flagella peritrichous flagella ----HH
antigens. antigens.
–Capsule – Capsule – KK antigen (antigen ( ViVi for Salmonella).for Salmonella).
–Cell envelope (wall)Cell envelope (wall)
–LPS (endotoxin) –LPS (endotoxin) –
OO antigen. antigen.
–various outer various outer
membrane proteins.membrane proteins.
–Pili Pili - various antigen - various antigen
types, some encoded types, some encoded by plasmids by plasmids

18. Pathogenesis and Immunity
Common virulence factors
Type III secretion systems: possessed by some Enterobacteriaceae
pathogens, e.g., E. coli, Yersinia, Salmonella, and Shigella; facilitate
transport of bacterial virulence factors directly into host cells.
Endotoxin (Lipid A of LPS)
Capsule
Antigenic phase variation
Acquisition of growth factors (e.g. Fe)
Resistance to serum killing
Antimicrobial resistance

19. HE Agar: Growth of Enteric Pathogens and Commensals

20. Escherichia coliEscherichia coli
Toxins: two types of enterotoxin; Shiga-type toxin;
Enteroaggregative ST-like toxin; Hemolysins;
Endotoxin
Virulence factors that protect the bacteria from host
defenses: Capsule/Iron capturing ability (enterochelin)

21. E. coli
May be hemolytic on CBA – more common in pathogenic
strains
KEY tests for the normal strain:
TSI is A/A + gas
LIA K/K
Urea –
Indole +
Citrate –
Motility +
There is an inactive biotype that is anaerogenic, lactose –,
and nonmotile.

22. Enteropathogenic Enteropathogenic E. coliE. coli
 fever
 infant diarrhea
 vomiting
 nausea
 non-bloody stools
 Destruction of surface microvilli loose attachment mediated by
bundle forming pili (Bfp);
 Stimulation of intracellular calcium level;
 rearrangement of intracellular actin,
Enterotoxigenic Enterotoxigenic E. coliE. coli
A watery diarrhea, nausea, abdominal cramps and low-grade
fever for 1-5 days.
Travellers diarrhea and diarrhea in children in developing
countries
Transmission is via contaminated food or water.

23. E.coli-E.coli-Enteroinvasive (EIEC)Enteroinvasive (EIEC)
The organism attaches to the intestinal mucosa via pili
Outer membrane proteins are involved in direct penetration,
invasion of the intestinal cells, and destruction of the intestinal
mucosa.
There is lateral movement of the organism from one cell to
adjacent cells.
Symptoms- fever,severe abdominal cramps, malaise, and watery
diarrhea followed by scanty stools containing blood, mucous,& pus.
resembles shigellosis
E.coli-E.coli-c. Enteropathogenic (EPEC)c. Enteropathogenic (EPEC)
Malaise and low grade fever diarrhea, vomiting, nausea, non-
bloody stools
Bundle forming pili are involved in attachment to the intestinal
mucosa.
This leads to changes in signal transduction in the cells,
effacement of the microvilli, and to intimate attachment via a non-
fimbrial adhesion called intimin.
This is a problem mainly in hospitalized infants and in day care
centers.

24. Enterohemorrhagic (EHEC) Enterohemorrhagic (EHEC)
Hemorrhagic
–bloody, copious diarrhea
–few leukocytes
–afebrile
hemolytic-uremic syndrome
–hemolytic anemia
–thrombocytopenia
(low platelets)
–kidney failure
• Usually O157:H7
Transmission electron micrograph

25. Enteroaggregative Enteroaggregative E. coli E. coli
a cause of persistent, watery diarrhea with
vomiting and dehydration in infants.
That is autoagglutination in a ‘stacked brick’
arrangement.
the bacteria adheres to the intestinal mucosa
and elaborates enterotoxins
(enteroaggregative heat-stable toxin, EAST).
The result is mucosal damage, secretion of
large amounts of mucus, and a secretory
diarrhea.

26. ShigellaShigella

27. Shigella species
Shigella
Contains four species that differ antigenically and, to a lesser
extent, biochemically.
S. dysenteriae (Group A)
S. flexneri (Group B)
S. boydii (Group C)
S. sonnei (Group D)
bacillary dysentery, shigellosis, bloody feces, intestinal pain, pus
Biochemistry
TSI K/A with NO gas
LIA K/A
Urea –
Motility -
All ferment mannitol except S. dysenteriae
S. sonnei may show delayed lactose fermentation

28. Shiga toxinShiga toxin
1. Chromosomally encoded
2. Neurotoxic
3. Enterotoxic
4. Cytotoxic
EnterotoxicityEnterotoxicity can make the disease clinically
appear as a diarrheadiarrhea.
The toxin inhibits protein synthesistoxin inhibits protein synthesis (acting on the
60S ribosome and lysing 28S rRNA).

29. Clinical significanceClinical significance
man only "reservoir"man only "reservoir"
mostly young children mostly young children
–fecal to oral contactfecal to oral contact
–children to adultschildren to adults
transmitted by adult food handlerstransmitted by adult food handlers
–unwashed handsunwashed hands

30. Diagnosis of Shigella infectionDiagnosis of Shigella infection
Specimen: stool.
Culture and Identification
Quick immunological methods:
1. Immunofluorescent “ball” test;
2. Coagglutination.

31. XLD Agar: Growth of Shigella and Proteus

32. Only a few types that are commonly associated
with characteristic human diseases -
1. 1. S. enteritidisS. enteritidis
2. 2. S. cholerae-suisS. cholerae-suis
3. 3. S. typhiS. typhi
SalmonellaSalmonella
several syndromes including gastroenteritis, enteric
(typhoid) fever or septicemia

33. Salmonella
Biochemistry
TSI K/A + gas and H
2
S: S. typhi produces only a small
amount of H
2
S and no gas , and S. paratyphi A produces
no H
2
S
LIA K/K with H
2
S with S. paratyphi A giving K/A results
Urea –
Motility +
Citrate +/-
Indole -
Virulence factors
Endotoxin – may play a role in intracellular survival
Capsule (for S. typhi and some strains of S. paratyphi)
Adhesions – both fimbrial and non-fimbrial

34. Salmonella typhiSalmonella typhi
• The organism is transmitted from:
1. a human reservoir 1. a human reservoir
2. in the water supply (if poor sanitary conditions)2. in the water supply (if poor sanitary conditions)
3. in contaminated food3. in contaminated food
The antigenic structures of salmonellae used in serologic typingThe antigenic structures of salmonellae used in serologic typing

35. Virulence factorsVirulence factors
Endotoxin – may play a role in intracellular survival
Capsule (for S. typhi and some strains of S. paratyphi)
Adhesions – both fimbrial and non-fimbrial
Type III secretion systems and effector molecules – 2 different systems may be
found:
–One type is involved in promoting entry into intestinal epithelial cells
–The other type is involved in the ability of Salmonella to survive inside macrophages
Outer membrane proteins - involved in the ability of Salmonella to survive
inside macrophages
Flagella – help bacteria to move through intestinal mucous
Enterotoxin - may be involved in gastroenteritis
Iron capturing ability

36. XLD Agar: Appearance of Salmonella

37. DDiagnosisiagnosis
A. SpecimensA. Specimens
 a) Enteric fever: blood, bone marrow, stool, a) Enteric fever: blood, bone marrow, stool,
urine.urine.
 b) Food poisoning: stool, vomitus, suspected food.b) Food poisoning: stool, vomitus, suspected food.
 c) Septicemia: blood. c) Septicemia: blood.
B. Culture and identificationB. Culture and identification
C. Widal testC. Widal test

38. Klebsiella
NF of GI tract, but potential pathogen in other areas
TSI A/A + gas
LIA K/K
Urea +
Citrate +
MR-, VP+
Motility -
Has both O and
K antigens

39. Klebsiella
Virulence factors
Capsule
Adhesions
Iron capturing ability
Clinical significance
Causes pneumonia, mostly in immunocompromised hosts.
 Permanent lung damage is a frequent occurrence (rare in
other types of bacterial pneumonia)
A major cause of nosocomial
infections such as septicemia and
meningitis
K.Pneumoniae On BA
K.Pneumoniae on BA and MAC

40. Proteus Proteus
General characteristics: “swarming” phenomenon General characteristics: “swarming” phenomenon
on nonselective agaron nonselective agar (P.vulgaris; P.mirabilis and (P.vulgaris; P.mirabilis and
P.myxofaciens) P.myxofaciens)
P.vulgaris strains (OX-19, OX-K, OX-2)have P.vulgaris strains (OX-19, OX-K, OX-2)have
common antigen with Rickettsia (Weil-Felix test). common antigen with Rickettsia (Weil-Felix test).

urinary tract infections; food poisoningurinary tract infections; food poisoning..

41. Proteus spp.
Some enteric bacteria are motile.
Klebsiella species are not motile,
while Proteus species move very
actively by means of peritrichous
flagella, resulting in "swarming"
on solid medium.
Some strains of E. coli produce
hemolysis on blood plates.

42. CITROBACTERCITROBACTER
Resident of soil, water, stool
• C.freundii
• UTI and bacteremia

43. SerratiaSerratia

44. Serratia
A free-living saprophyte
TSI A/A or K/A; +/- gas (does not ferment lactose)
LIA usually K/K
Citrate +
Motility +
Urea +/-
Has been found in RT and UT infections
Is resistant to many antimicrobics

45. Special Features:
Used in the differentiation of
genera and species. e.g. E.
coli (+) from Klebsiella (-).
Positive test
e.g. E. coli
Negative test
e.g. Klebsiella
IIndole testndole test
Positive
Klebsiella,
Enterobacter
Negative
E. coli
Citrate Utilization Test
Urease Test
Positive
test
Negative test

46. MMR/R/VVP testP test
Results
Methyl Red test Voges-Proskauer test
Red: Positive MR (E. coli)
Yellow or orange: Negative MR (Klebsiella)
Pink: Positive VP (Klebsiella)
No pink: Negative VP (E. coli)

47. Gram Gram
stainstain
OxidaseOxidase Nitrate Nitrate
reductasereductase
O/FO/F MacConMacCon
keykey
SSSS EMBEMB
E. coliE. coli -ve rod-ve rod -ve-ve +ve+ve O+/F+O+/F+ LFLF LFLF Metallic Metallic
sheensheen
CitrobacterCitrobacter-ve rods-ve rods-ve-ve +ve+ve O+/F+O+/F+ LFLF LFLF DarkDark
KlebsiellaKlebsiella-ve rods-ve rods-ve-ve +ve+ve O+/F+O+/F+ LFLF LFLF DarkDark
EnterobacterEnterobacter-ve rods-ve rods-ve-ve +ve+ve O+/F+O+/F+ LFLF LFLF DarkDark
SalmonellaSalmonella-ve rods-ve rods-ve-ve +ve+ve O+/F+O+/F+ NLFNLF NLF/NLF/
H2SH2S
ColorlessColorless
ShigellaShigella -ve rods-ve rods-ve-ve +ve+ve O+/F+O+/F+ NLFNLF NLFNLFColorlessColorless
ProteusProteus -ve rods-ve rods-ve-ve +ve+ve O+/F+O+/F+ NLFNLF NLF/NLF/
H2SH2S
ColorlessColorless
Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae

48. TSITSI IndoleIndoleMRMR VPVP CitrateCitrateUreaseUreaseMotilityMotility
E. coliE. coli A/A/-A/A/-+ve+ve +ve+ve -ve-ve -ve-ve -ve-ve MotileMotile
Citrobacter Citrobacter
freundiifreundii
A/A/-A/A/-+ve+ve +ve+ve -ve-ve +ve+ve -ve-ve MotileMotile
Klebsiella Klebsiella
pneumoniaepneumoniae
A/A/-A/A/--ve-ve -ve-ve +ve+ve +ve+ve +ve+ve Non Non
motilemotile
Enterobacter Enterobacter
cloacaecloacae
A/A/-A/A/--ve-ve -ve-ve +ve+ve +ve+ve +ve+ve MotileMotile
Salmonella Salmonella
typhityphi
A/Alk/+A/Alk/+-ve-ve +ve+ve -ve-ve +ve+ve -ve-ve MotileMotile
Shigella Shigella
boydiiboydii
A/Alk/-A/Alk/--ve-ve +ve+ve -ve-ve -ve-ve -ve-ve Non Non
motilemotile
ProteusProteus
mirabilismirabilis
A/Alk/+A/Alk/+-ve-ve +ve+ve -ve-ve +ve+ve +ve+ve MotileMotile
SwarwingSwarwing
Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae

49. Oxidase TestOxidase Test
Positive
Negative
Pseudomonas

Enterobacteriacea
e
 Nitrate test: +ve further
reduction to N
2
 Growth on cetrimide agar:
Pale colonies with green
pigmentation
MacConkey’s agar & TSI
Lactose fermenter
Pink colonies on MacConkey
& acidic butt and slant on TSI
colorless colonies on MacConkey
& acidic butt alkaline slant onTSI
Lactose non-fermenter
IMV
i
C test
& EMB
IMV
i
C
++ - -
& black colonies
with metalic
shines on EMB
E.coli
IMV
i
C
- - ++
Klebsiella
No H
2
S production
(no blacking in TSI)
H
2
S production
(blacking in TSI)
Shigella
Urease production
+ve
Proteus
-ve
SS agar
colorless colonies with black centers
Salmonella
 O/F test: O
+
/F
-
Motility
Not motile
Motile
Enterobacter