ENTEROBACTERIACEAE for Students in Microbiology by Dr.T.V.Rao MD

1. ENTEROBACTERIACEA
E
basics for students
Dr.T.V.Rao MD
01/28/18 Dr.T.V.Rao MD 1

2. Bacteria
Gram positive Gram negative
Cocci Bacilli Cocci Rods
01/28/18 Dr.T.V.Rao MD 2

3. Enterobacteriaceae
• Commonly present in large intestine
• Non sporing , Non Acid fast, Gram –
bacilli.
• A complex family of organisms,
• Some are non pathogenic
• A few are highly Pathogenic,
• Some commensals turn out to be
pathogenic. as in UTI after catheterization.
01/28/18 Dr.T.V.Rao MD 3

4. 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)
01/28/18 Dr.T.V.Rao MD 4

5. Basic Tests ……
• Basic characters
• Catalase +
• Oxidase -
• Reduce nitrates,
• All are Gram negative and non spore
forming.
• Wide diversity / Antigenic heterogeneity,
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6. Classification of Enterobacteriaceae
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
01/28/18 Dr.T.V.Rao MD 6

7. Enterobacteriaceae: Major
Genera
• Escherichia
• Shigella
• Salmonella
• Edwardsiella
• Citrobacter
• Yersinia
• Klebsiella
• Enterobacter
• Serratia
• Proteus
• Morganella
• Providencia

8. Basic Classification
• I Lactose fermenters E.coli, Escherichia
Klebsiella,
• II Late Lactose fermenters, Shigella
sonnei,
• III Non Lactose fermenters, Salmonella
Shigella,
Commonly tested with MacConkey medium
Many are commensals Lactose fermenters
Also called as coli forms , Enteric Bacilli
01/28/18 Dr.T.V.Rao MD 8

9. Enterobacteriaceae
Taxonomical
• Tribe I Escherichia
Genus – 1 Escherichia,
2 Edwardsville
3 Citrobacter
4 Salmonella
5 Shigella
01/28/18 Dr.T.V.Rao MD 9

10. Enterobacteriaceae.
• Tribe II; Klebsiella
Genus 1. Klebsiella
2.Enterobacter,
3.Hafnia
4 Serratia
Tribe III ; Proteeae
Genus
1,Proteus
2,Morganella
3 . Providencia
01/28/18 Dr.T.V.Rao MD 10

11. Classification of
Enterobacteriaceae ( Contd )
• Tribe IV;
Erwinieae
Genus 1.
Erwinia
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12. Identification of Enterobacteriaceae
Differentiation between LF & NLF by Growth on MacConkey agar
• Method:
– MacConkey agar is inoculated with tested organism
using streak plate technique
– Incubate the plate in incubator at 37 C/24 hrs
• Results:
– LF organism appears as pink colonies (e.g. E. coli)
– NLF organism appears as colorless colonies (e.g.
Shigella)
Flame & Cool
Flame & Cool
Flame & Cool
1 2
3
4
5
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13. Enterobacteriaceae on
different media
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14. Highly Pathogenic
Enterobacteriaceae
• Salmonella
• Shigella
• All are Lactose – non fermenters,
• Produce colorless colonies on
MacConkey medium
• LF also called as Para colons,
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15. 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.
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16. Determinants of pathogenicity
• Major antigens
- K (“kapsel”) Ag – associated with polysaccharide
capsules, predominant in Klebsiella
- H (“hauch”, breath) Ag – flagellar antigens,
describe swarming colonies, absent in non-motile,
impt. for Salmonella serotyping
- O (“ohne hauch”, w/o breath) Ag – somatic
antigens, nonmotile, nonflagellate bacteria

17. 01/28/18 Dr.T.V.Rao MD 17

18. 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.
01/28/18 Dr.T.V.Rao MD 18

19. E.coli
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.
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20. E.coli on Blood Agar
• Many
pathogenic
strains are
haemolytic
on blood
agar.
01/28/18 Dr.T.V.Rao MD 20

21. E.coli
Biochemical Characters,
• Glucose,Lactose,Mannitol,Maltose
fermented. with A/G
Indole +
Methyl Red +
Voges Proskauer – I,M,Vi,C tests.
Citrate –
Urease not produced.
01/28/18 Dr.T.V.Rao MD 21

22. E.coli
Antigenic Structure
• Somatic 0 170
• Capsular K 100
• Flagella H 75
• Virulence factors
Surface Antigens Toxins
O Endotoxic activity
K protects against the phagocytosis
Fimbriae promote virulence ( important in UTI )
01/28/18 Dr.T.V.Rao MD 22

23. Virulence Factors
• Two types of virulence factors in
E.coli
• Surface antigens and Toxins
• The somatic lipopolysaccharide
surface O antigen has endotoxic
activity and protects from
phagocytosis and bactericidal effects
of complement .
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24. Determinants of pathogenicity
• Enterotoxins (ETEC)
– Heat-labile toxins (LT-I, LT-II)
– Heat-stable toxins (ST)
• Hemolysins – from enterohemorrhagic
(verotoxin-producing) E. Coli
 most virulent uropathogenic strains of E. coli
• Antiphagocytic capsule – from K1 strain of E.
Coli (80% of neonatal meningitis)

25. Fimbriae
• Fimbriae also promote virulence
• Present in large numbers causing
mannose sensitive Haemagglutination
• Colonisation factor antigens is
enterotoxigenic E.coli
• E.coli produce two kinds of Exotoxins
hemolysins and enterotoxins
01/28/18 Dr.T.V.Rao MD 25

26. Toxins
and E.coli
• E.coli produce Exotoxins
• Hemolysins, Enterotoxins causes
Diarrheas,
• Important toxins produces.
• Heat labile HL Heat stable HS
Vero toxins VT Like Shigella
toxins01/28/18 Dr.T.V.Rao MD 26

27. 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:
01/28/18 Dr.T.V.Rao MD 27

28. Toxins
• LT – is heat labile and binds to
specific Gm1 gangliosides on the
epithelial cells of the small
intestine where it ADP-ribosylates
Gs which stimulates adenylate
cyclase to increase production of
cAMP.
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29. Toxins
• Increased cAMP alters the
activity of sodium and
chloride transporters
producing an ion imbalance
that results in fluid transport
into the bowel.
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30. Toxins in E.coli
• Produce Enterotoxin L T and S T
• Labile toxin 1956 De experiments in
Rabbit ileal loop causes outpouring of
fluids
• E.coli Labile toxin like Cholera toxin
• L T contains component A and B
• A = Active B= Binding
• B causes Binding with Gm I Ganglioside
receptor on Intestinal epithelial cells
01/28/18 Dr.T.V.Rao MD 30

31. E. coli toxins
• Both enterotoxins
are composed of
five beta subunits
(for binding) and
1 alpha subunit
(has the toxic
enzymatic
activity).
01/28/18 Dr.T.V.Rao MD 31

32. Toxins E.coli
Labile toxin
• Component A Activated to A1 and A2
• A1 Activates adenyl cyclase in the
enterocytes to form cyclic adenosine
5 monophosphate
• Causes to increase outflow of water
and electrolytes in the gut lumen
causes Diarrhea
01/28/18 Dr.T.V.Rao MD 32

33. Toxins of E.coli
Stable Toxin
• ST A and ST B
• ST A Acts by activation of Cyclic
guano sine monophosphate.( C GMP )
• Causes fluid accumulation in Intestine.
• E.coli ( Some ) produce
Verocytotoxin causes cytotoxicity
to Vero cells.
• Acts like Shigella dysentery toxin
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34. E. coli infections
• Neonatal meningitis – is the leading cause of
neonatal meningitis and septicemia with a high
mortality rate.
– Usually caused by strains with the K1 capsular antigen.
• Gastroenteritis – there are several distinct types of E.
coli that are involved in different types of
gastroenteritis:
– enterotoxigenic E. coli (ETEC),
– enteroinvasive E. coli (EIEC),
– enteropathogenic E. coli (EPEC) ,
– enteroaggregative E. coli (EAEC), and
– enterohemorrhagic E. coli (EHEC).
01/28/18 Dr.T.V.Rao MD 34

35. Classification of E.coli
1.Enteropathogenic EPEC
2.Enterotoxigenic ETEC
3.Enteroinvasive EIEC
4.Enterohemorrhagic EHEC
5.Enteroaggresive EAEC
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36. Enteropathogenic E.coli
• Causes diarrheal disease in children,
• EPEC O26/O11
• Produce Verocytotoxin
• Infantile enteritis, Involves upper part of Intestine
• Brush border of the intestine is lost
• Intimacin – EPEC adhesion factor.
• Frequent in summer months
• Poor hygiene predisposes.
• Out breaks in Institutions
01/28/18 Dr.T.V.Rao MD 36

37. Enteropathogenic E.coli
• Causes diarrheal disease in children,
• EPEC O26/O11
• Produce Verocytotoxin
• Infantile enteritis, Involves upper part of
Intestine
• Brush border of the intestine is lost
• Intimacin – EPEC adhesion factor.
• Frequent in summer months
• Poor hygiene predisposes.
• Out breaks in Institutions
Dr.T.V.Rao MD 3701/28/18

38. EPEC
• EPEC are identified by serotyping
by their O and B antigens
• Diagnosis is difficult during
sporadics
• Routine culture is done for
isolation
• The EPEC fails to ferment sorbitol
• Causes the disruption of brush
border
01/28/18 Dr.T.V.Rao MD 38

39. Laboratory Diagnosis
EPEC
• Confirm with Polyvalent
sera
• Test Sero groups with
polyvalent and monovalent
sera.
• HEp2 – adherence.01/28/18 Dr.T.V.Rao MD 39

40. Enterotoxigenic E.coli
• Common and causes endemics in
developing countries in all age
groups
• May be mild watery diarrhoea to
fatal conditions
• Fimbrial colonization factor
antigens
01/28/18 Dr.T.V.Rao MD 40

41. Enterotoxigenic E.coli
• Produce Heat stable /Heat labile toxins
• Adheres to epithelium of small intestine.
• Present with Nausea, Vomiting and Lose stool
• H L like cholera toxin
• Causes accumulation of fluids
• Adhesive factors
Fimbriae specific receptor in the intestinal
epithelium CFA
Mortality in children < 5 years
01/28/18 Dr.T.V.Rao MD 41

42. ETEC
• Causes travelers diarrhea
• Water contaminated with Human and Animal
feces predisposes.
• Laboratory Diagnosis
Demonstration of Enterotoxin LT and ST
Tissue culture tests,
ELISA
Passive agglutination tests.
Animal experiments in Rabbit ileal loop test.
01/28/18 Dr.T.V.Rao MD 42

43. Treatment and Prophylaxis
in Travelers diarrhea
• Doxycycline,
• Trimethoprim,
• Norfloxacillin
• Fluroquinolones
• Avoid contaminated food,
• Safe protected water ,prefer bottled
water,
• Hot foods, Hot Drinks,
• Boiled milk
Dr.T.V.Rao MD 4301/28/18

44. Treatment and Prophylaxis
in Travelers diarrhea
• Doxycycline,
• Trimethoprim,
• Norfloxacillin
• Fluroquinolones
• Avoid contaminated food,
• Safe protected water ,prefer bottled water,
• Hot foods, Hot Drinks,
• Boiled milk
01/28/18 Dr.T.V.Rao MD 44

45. Enteroinvasive E.coli
• Resembles Shigella in many respects
• Non lactose fermenter and non motile
• They invade the intestinal epithelium
• Penetrate HeLa cells in tissue culture
• May produce mild diarrhoea to frank
dysentery
• Sereny test
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46. Entero invasive E.coli
• Some are non motile
strains,
• Atypical resembles
like Shigella.
• Clinically mild
diarrhea
• Sereny test positive
animal Rabbit.
• ELISA
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47. Enterohemorrhagic E.coli
• Produce Verocytotoxin or shiga like toxin
• Mild diarrhea - can be fatal hemorrhagic
colitis. and uremic syndrome.
• Present in Human and Animal feces.
• Hemorrhagic complication with O157 in
Japan and USA.
• Salads vegetables, Radish Proper cooking
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48. Dr.T.V.Rao MD 4801/28/18

49. Enterohemorrhagic E.coli
• The primary target for VT appear to be
vascular endothelial cells
• This may contribute to HUS with
characteristic renal lesion is capillary
micro angiopathy
• The typical EHEC serotype is 0 157 ; H7
• The disease may manifest as food
poisoning
• Occurs due to contamination of feces of
humans or animals
01/28/18 Dr.T.V.Rao MD 49

50. Enterohemorrhagic E.coli can
cause HUS
• HUS develops when the toxin from E. coli
bacteria, known as Shiga-like toxin (SLT)
enters the circulation by binding to
special receptors. These Shiga-toxin
receptors, known as Gb3 receptors , are
probably heterogeneously distributed in the
major body organs allowing disparate
thrombotic (blood clotting) impacts in different
HUS victims, although the greatest receptor
concentration appears to be in the kidneys,
especially in children.
Dr.T.V.Rao MD 5001/28/18

51. EHEC ( E.coli )
• Culture
• DNA detection methods.
• Cytotoxic effects on Vero
cells.
• Detection with monovalent
sera O157/H701/28/18 Dr.T.V.Rao MD 51

52. Enteroaggresive E.coli
• They appear aggregated in s
stacked brick formation Hep-2 cell
• They produce persistent diarrheal
• They produce weight heat stable
enterotoxin called as low
molecular heat stable enterotoxin
01/28/18 Dr.T.V.Rao MD 52

53. Enteroaggresive E.coli
EAEC
• Stool
Culture
methods
• Detection of
Enterotoxin
01/28/18 Dr.T.V.Rao MD 53

54. Enterobacteriaceae: Wound
Infection, Bacteremia
• Wound Infection: Escherichia
coli, Enterobacter spp.,
Klebsiella pneumoniae, and
Proteus mirabilis
• Bacteremia: Escherichia coli,
Enterobacter spp., Klebsiella
pneumoniae, and Proteus
mirabilis

55. 55
Treatment – E.coliTreatment – E.coli
Gastrointestinal diseaseGastrointestinal disease
• Fluid replacement
• Antibiotics
– not used usually unless
systemic infections
prevails
–e.g. hemolytic-uremia
syndrome01/28/18 Dr.T.V.Rao MD

56. E. coli
– Clinical
significance
• Is the leading
cause of urinary
tract infections
which can lead
to acute cystitis
(bladder
infection) and
pyelonephritis
(kidney
infection).
01/28/18 Dr.T.V.Rao MD 56

57. Ascending urinary tractAscending urinary tract
infectioninfection
01/28/18 Dr.T.V.Rao MD 57

58. Urinary Tract Infections
• E.coli produce urinary tract infection.
• Majority of UTI s are produce by E.coli.
• Instrumentation, Prostatic enlargement,
Urinary caliculi,Pregnancy,
• Asymptomatic Bacteriuria in pregnant
women,
• Pyelonephritis,
01/28/18 Dr.T.V.Rao MD 58

59. Facts on UTI
• Women suffer more than males Short urethra
Pregnancy, Sexual intercourse /Honey moon
cystitis.
• Other factors
Urethral obstruction,
Urinary stones
Congenital malformation's
Neurological disorders,
Catheterization , Cystoscopy
Usually cystitis is produced from fecal strains
entering urethra
01/28/18 Dr.T.V.Rao MD 59

60. Culturing for E.coli
• Mid stream sample/semi quantitative
culturing (Kass et al ) >_ 1.00,000/ml of
urine. ( significant Bacteriuria )
• Urine should not be kept in wards for > 2
hours and to be preserved at 4 c
• Culture by standard loop method.
• Fixed volume cultured on MacConkey
agar Lactose fermenters I M Vi C
• Antibiotic sensitivity tested.
01/28/18 Dr.T.V.Rao MD 60

61. Other Bacteria and UTI
• Majority of UTI are caused by E.coli
• Other agents which cause UTI,
1 Staphylococcus
2.Proteus.
3.Pseudomonas,
4.Klebsilella spp,Citrobacter,
5.Enteococcus.
01/28/18 Dr.T.V.Rao MD 61

62. Other infection with
E.coli
• Pyogenic infections.
• Intraabdominal infections
• Peritonitis. Abscess.
• Septicemias
• Produce Drug resistant
infections.
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63. 01/28/18 Dr.T.V.Rao MD 63

64. Klebsiella, Enterobacter,
Serratia & Hafnia sp.
• Usually found in intestinal tract
• Wide variety of infections, primarily pneumonia,
wound, and UTI
• General characteristics:
– Some species are non-motile
– Simmons citrate positive
– H2S negative
– Phenylalanine deaminase negative
– Some weakly urease positive
– MR negative; VP positive
01/28/18 Dr.T.V.Rao MD 64

65. Klebsiella species
• Usually found in GI tract
• Four major species
• K. pneumoniae is mostly commonly isolated
species
– Possesses a polysaccharide capsule, which
protects against phagocytosis and
antibiotics AND makes the colonies moist
and mucoid
– Has a distinctive “yeasty” odor
– Frequent cause of nosocomial pneumonia
01/28/18 Dr.T.V.Rao MD 65

66. Klebsiella
• Klebsiella is a genus of non-motile,
Gram-negative, oxidase-negative, rod-
shaped bacteria with a prominent
polysaccharide-based capsule. It is
named after the German microbiologist
Edwin Kleb's (1834–1913). Frequent
human pathogens, Klebsiella organisms
can lead to a wide range of disease
states, notably pneumonia, urinary tract
infections, septicaemia, and soft tissue01/28/18 Dr.T.V.Rao MD 66

67. Klebsiella
• The genus was originally divided into 3
main species based on biochemical
reactions. Today, 7 species with
demonstrated similarities in DNA
homology are known. These are (1)
Klebsiella pneumoniae, (2) Klebsiella
ozaenae, (3) Klebsiella rhinoscleromatis,
(4) Klebsiella oxytoca, (5) Klebsiella
planticola, (6) Klebsiella terrigena, and (7)
Klebsiella ornithinolytica01/28/18 Dr.T.V.Rao MD 67

68. Klebsiella
• K pneumoniae is the most medically
important species of the group. K
oxytoca and K rhinoscleromatis have
also been demonstrated in human
clinical specimens. In recent years,
klebsiellae have become important
pathogens in nosocomial infections.]
01/28/18 Dr.T.V.Rao MD 68

69. Klebsiella
• 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
01/28/18 Dr.T.V.Rao MD 69

70. Klebsiella species
• Usually found in GI tract
• Four major species
• K. pneumoniae is mostly commonly isolated
species
– Possesses a polysaccharide capsule, which
protects against phagocytosis and
antibiotics AND makes the colonies moist
and mucoid
– Has a distinctive “yeasty” odor
– Frequent cause of nosocomial pneumonia
01/28/18 Dr.T.V.Rao MD 70

71. Klebsiella species (cont’d)
– Significant biochemical reactions
• Lactose positive
• Most are urease positive
• Non-motile
01/28/18 Dr.T.V.Rao MD 71

72. 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
01/28/18 Dr.T.V.Rao MD 72

73. Enterobacter species
• Comprised of 12 species; E. cloacae and
E. aerogenes are most common
• Isolated from wounds, urine, blood and
CSF
• Major characteristics
– Colonies resemble Klebsiella
– Motile
– MR negative; VP positive
01/28/18 Dr.T.V.Rao MD 73

74. Serratia species
• Seven species, but S. marcescens is the
only one clinically important
• Frequently found in nosocomial
infections of urinary or respiratory
tracts
• Implicated in bacteremic outbreaks in
nurseries, cardiac surgery, and burn
units
• Fairly resistant to antibiotics
01/28/18 Dr.T.V.Rao MD 74

75. Serratia species (cont’d)
• Major characteristics
– Ferments lactose slowly
– Produce characteristic pink pigment,
especially when cultures are left at room
temperature
S. marscens on
nutrient agar →
01/28/18 Dr.T.V.Rao MD 75

76. Hafnia species
• Hafnia alvei is only species
• Has been isolated from many
anatomical sites in humans and the
environment
• Occasionally isolated from stools
• Delayed citrate reaction is major
characteristic
01/28/18 Dr.T.V.Rao MD 76

77. Other Enterobacteriaceae
• Proteus, Providencia, and Morganella
– Are all part of the NF of the GI tract (except
Providencia).
– All motile, with Proteus swarming
– PA +
– Lysine deamination + (LIA R/A)
– Urea + for most, strongly + for Proteus
– TSI variable (know the reactions for each in
the lab!)
– Indole – only P. mirabilis is -
01/28/18 Dr.T.V.Rao MD 77

78. Proteus, Morganella &
Providencia species
• All are normal intestinal
flora
• Opportunistic pathogens
• Deaminate phenylalanine
• All are lactose negative
01/28/18 Dr.T.V.Rao MD 78

79. Proteus species
• P. mirabilis and P. vulgaris are widely recognized
human pathogens
• Isolated from urine, wounds, and ear and
bacteremic infections
• Both produce swarming colonies on non-selective
media and have a distinctive “burned chocolate”
odor
• Both are strongly urease positive
• Both are phenylalanine deaminase positive
01/28/18 Dr.T.V.Rao MD 79

80. Proteus species (cont’d)
• A exhibits characteristic “swarming”
• B shows urease positive on right
01/28/18 Dr.T.V.Rao MD 80

81. Morganella species
• Morganella morganii is only
species
• Documented cause of UTI
• Isolated from other anatomical
sites
• Urease positive
• Phenylalanine deaminase positive01/28/18 Dr.T.V.Rao MD 81

82. Providencia species
• Providencia rettgeri is pathogen of
urinary tract and has caused nosocomial
outbreaks
• Providenicia stuartii can cause
nosocomial outbreaks in burn units and
has been isolated from urine
• Both are phenylalanine deaminase
positive
01/28/18 Dr.T.V.Rao MD 82

83. Citrobacter species
• Citrobacter freundii associated
with nosocomial infections (UTI,
pneumonias, and intraabdominal
abscesses)
• Ferments lactose and hydrolyzes
urea slowly
• Resembles Salmonella sp.
01/28/18 Dr.T.V.Rao MD 83

84. • Program created by Dr.T.V.Rao MD for
basic learning on Enterobacteriaceae for
Microbiology Students
• Email
• doctortvrao@gmail.com
01/28/18 Dr.T.V.Rao MD 84