Enterobacteriaceae

1. Chapter 9
ENTEROBACTERIACEAE & 19

2. OBJECTIVES
 Describe the general characteristics of the Enterobacteriaceae family.
 Discuss the principle, purpose, and interpretation of the following tests:
 Citra te
 DN a se
 L ysine iron a ga r
 Methyl R ed
 MIO
 N itra te
 ON PG
 Oxida se
 Phenyl a nil i n e dea mina se
 TSI
 Urea se
 Voges - Pra sk a uer
 Distinguish between lactose fermenting and non -lactose fermenting Enterobacteriaceae on the
MacConkey, Eosin Methylene Blue, Hektoen, and XLD agars.
 List the non-motile Enterobacteriaceae.
 L i s t t h e E n t e r o b a c t e r i a c e a e t h a t p r o d u c e H 2S .
 Differentiate the genera of Enterobacteriaceae based on biochemical tests.
 Explain the common modes of transmission and clinical significance of Salmonella, Shigella and E.
coli 0157:H7.
 Differentiate the following species:
 E.coli and E. coli 0157:H7
 Proteus vulgaris and Proteus mirabilis
 Klebsiella pneumoniae and Klebsiella oxytoca.
 Discuss the clinical significance of the Enterobacteriaceae in this unit.

3. TAXONOMY
 Family
 Enterobacteriaceae
 Genera
 Escherichia  Serratia
 Shigella  Proteus
 Edwardsiella  Morganella
 Citrobacter  Providencia
 Klebsiella  Yersinia
 Enterobacter  Salmonella

4. GENERAL CHARACTERISTICS
 Found in nature
 Plants
 Soil
 Water
 Intestines of humans
 Most are normal enteric flora

5. BIOCHEMICAL CHARACTERISTICS
 Ferment glucose
 Oxidase negative
 Reduce nitrate to nitrite
Oxidase Test Nitrate Reduction
Glucose Fermentation

6. MICROSCOPIC CHARACTERISTICS
 Gram negative bacilli
 Most are motile
 Non-spore forming

7. GROWTH REQUIREMENTS
 Facultative anaerobes
 Incubation length
 18-24 hours
 Incubation temperature
 35-37°C
 Incubation atmosphere
 Air

8. MACROSCOPIC CHARACTERISTICS
 Grow well on BAP
 Grow on enteric media
 MAC
 HE
 XLD
 EMB

9. THE SCOOP ON POOP!
 Enterobacteriaceae are routinely isolated from stool cultures
 ID work-up
 Only performed on those that are pathogens

10. ENTERIC MEDIA

11. BLOOD AGAR PLATE - 1
All Enterics grow on
BAP
Allows for
observation of colony
“true” morphology

12. BLOOD AGAR PLATE - 2

13. MACCONKEY (MAC) - 1
Selective
 Gram negative
Differential
 Lactose fermentation
 Fermenter
 PINK
 Non-fermenter
 Clear

14. MACCONKEY (MAC) - 2

15. XYLOSE-LYSINE-DESOXYCHOLATE (XLD) - 1
Selective
 Bile Salts
Differential
 Lactose fermentation
 YELLOW
 H2S
 BLACK

16. XYLOSE-LYSINE-DESOXYCHOLATE (XLD) - 2
Lactose Non-Fermenter; H2S Producer Lactose Fermenter

17. EOSIN-METHYLENE BLUE (EMB) - 1
 Selective
 Gram negative
 Differential
 Lactose fermentation
 BLUE-BLACK

18. EOSIN-METHYLENE BLUE (EMB) - 2

19. HEKTOEN ENTERIC (HE) - 1
Selective
 Gram negative
Differential
 Lactose fermentation
 YELLOW/ORANGE
 SALMON (Pink)
 H2S
 BLACK

20. HEKTOEN ENTERIC (HE)

21. BIOCHEMICAL REACTIONS

22. LACTOSE FERMENTATION TESTS
 Lactose non-fermenters
 Cannot break down lactose
 Lactose fermenters
 Have enzymes that allow for transport of lactose into the cell for
utilization
 Slow lactose fermenters
 Lack beta-galactosidase permease
 But have beta-galactosidase

23. TRIPLE SUGAR IRON AGAR (TSI) - 1
Media composition
 Sugars (Glucose, Lactose, Sucrose)
 Peptone - nutrient and energy
 pH indicator
 H 2 S indicator

24. TSI - 2
 How to read the tube?
 FIRST - Slant reaction
 SECOND – Butt reaction
 THIRD - H 2 S production
 “/” separates the reactions
 Circling the butt reaction denotes gas production
 Rules for test interpretation
 Glucose is always the preferred sugar for fermenters
 Glucose fermentation results in acid production
 Peptone utilization results in alkaline by-products

25. TSI REACTIONS - 1

26. TSI REACTIONS - 2

27. TSI REACTIONS - 3

28. TSI REACTIONS - 4

29. TSI REACTIONS - 5

30. TRIPLE SUGAR IRON AGAR (TSI)
Test Your Knowledge

31. O-F TESTS
 See power point presentation for chapter 21.

32. INDOLE
Tryptophanase + tryptophan → pyurvic acid + ammonia + indole
indole + 1% paradimethylaminocinnamaldehyde → color change (pink color)

33. UREA
Urease + urea (broth or agar) → ammonia + water + CO2
Ammonia produces a pH change in the media = Pink

34. UREASE
 Tube 1: Control
 Tube 2: Proteus vulgaris
 = Positive
 Tube 3: E. coli
 = Negative

35. CITRATE
+ = +

36. DNASE
Positive
 Escherichia coli (top):
negative-no clearing
 Serratia marcescens (bottom):
positive-clearing

37. DECARBOXYLASE TESTS - ORNITHINE
Control Positive Negative Control

38. DECARBOXYLASE TESTS – ARGININE &
LYSINE

39. METHYL RED
Glucose → Pyruvic acid → Mixed acid fermentation (pH 4.4)
Acid end-products detected with methyl red indicator

40. VOGES-PROSKAUER
Glucose → Pyruvic acid →Acetoin → Diacetyl
Diacetyl + KOH + α-naphthol → Red complex

41. ONPG
 Lactose fermenter have the β-
galactosidase enzyme, which
hydrolyzes ONPG into galactose
and orthonitrophenol = Color
change from colorless to yellow
 Non-lactose fermenters - do
not have the beta-galactosidase
enzyme to hydrolyze the ONPG
= No color change

42. MOTILITY
Motility is defined as
any growth away
from the stab
 1 = Non-motile
 2 = Motile
 3 = Motile

43. MOTILITY/INDOLE/ORNITHINE (MIO)
Indole Reaction
Motility Reaction
Ornithine Reaction

44. PHENYLALANINE DEAMINASE (PDA)
 Organisms that produce the
enzyme deaminase are able to
removes the amine group from
the amino acid phenylalanine
and releases the amine group as
free ammonia. As a result of
this reaction, phenylpyruvic
acid is also produced. Negative
 After incubation, 10% ferric
chloride is added to the media. Positive
 If phenylpyruvic acid was produced, it
will react with the ferric chloride and
turn dark green.
 If the medium remains a straw color,
the organism is negative for
phenylalanine deaminase production.

45. NITRATE
Add Zinc dust
= =
+ ? = =
?
Nitrate → Nitrite
Nitrite + sulfanilic acid + N,N-Dimethyl-1-naphthlamine → red

46. NITRATE
Positive
Positive
Negative

47. GELATIN HYDROLYSIS
 Escherichia coli: negative
(gelatin is solid)
 Pseudomonas aeruginosa: positive (gelatin has been liquefied)

48. ANALYTICAL PROFILE INDEX (API)

49. API 20-E

50. Negative Tests
Positive Tests

51. OPPORTUNISTIC
ENTERICS

52. ESCHERICHIA COLI

53. CLINICAL SIGNIFICANCE
 Most common human pathogen
 UTI
 Septicemia
 Meningitis
 Pneumoniae
 Endocarditis

54. COLONY MORPHOLOGY
BAP
MACHE
XLD
EMB

55. MICROSCOPIC EVALUATION
Gram negative
bacilli
Bipolar staining

56. BIOCHEMICAL IDENTIFICATION
Lactose fermenter
Gas from Glucose
IMV:C (++--)
 Indole – Positive
 MR – Positive
 VP – Negative
 Citrate – Negative
 ONPG – Positive
 Lysine - Positive

57. E. COLI Serotypes

58. ENTEROHEMMORAGIC E. COLI
(EHEC)
 E. coli 0157:H7
 Hemorrhagic colitis
 Hemolytic Uremic Syndrome (HUS)
 Low platelets
 Hemolytic anemia
 Kidney failure

59. ENTEROHEMMORAGIC E. COLI
(EHEC)
MacConkey with
Sorbitol
 Negative = clear/colorless
 Positive = PINK

60. KLEBSIELLA PNEUMONIAE

61. CLINICAL SIGNIFICANCE
 Most common isolate
 Lower respiratory infections in hospitalized patients
 Pneumonia
 Septicemia
 Meningitis
 UTI

62. COLONY MORPHOLOGY
 Very mucoid (wet) looking
 Due to a polysaccharide capsule

63. BIOCHEMICAL IDENTIFICATION
 Lactose fermenter
 Non-motile
 IMV:C (+-++); (--++)
 Indole
 K. oxytoca = Positive
 K. pneumoniae – Negative
 MR = Negative
 VP = Positive
 Citrate = Positive
 Urea = Positive
 DNase = Negative
 Ornithine = Negative (few + reported)

64. ENTEROBACTER

65. CLINICAL SIGNIFICANCE
 Opportunistic pathogens
 E. aerogenes
 E. cloacae – bacteremia and wound infections
 Meningitis
 Endocarditis
 Urinary tract infection
 Lower respiratory tract infections

66. BIOCHEMICAL IDENTIFICATION
 Lactose = Positive
 Motile
 IMV:C (--++)
 Indole = Negative
 MR = Negative
 VP = Positive
 Citrate = Positive
 Ornithine = Positive

67. SERRATIA SPECIES

68. CLINICAL SIGNIFICANCE
 Nosocomial
 UTI
 Respiratory infections
 Septicemia
 Endocarditis

69. COLONY MORPHOLOGY
Red pigmentation
 S. rubidea
 S. marcescens
Smells like peeled
potatoes
 S. odorifera

70. BIOCHEMICAL IDENTIFICATION
 Lactose = Negative
 IMV:C (-+++)
 Indole = Negative
 MR = Positive
 VP = Positive
 Citrate = Positive

71. PROTEUS SPECIES

72. CLINICAL SIGNIFICANCE
 Nosocomial infections
 UTIs
 Wound infections
 Septicemia
 Pneumonia

73. COLONY MORPHOLOGY
 Swarm on BAP
 Smells like burnt chocolate

74. BIOCHEMICAL IDENTIFICATION
 Lactose negative
 PDA - Positive
 Urease – Positive
 H 2 S – Positive (most)

75. SPECIES DIFFERENTIATION
 P. mirabilis
 Indole = Negative
 Ornithine = Positive
 P. vulgaris
 Indole = Positive
 Ornithine = Negative

76. MORGANELLA SPECIES

77. CLINICAL SIGNIFICANCE
 Nosocomial infections
 UTIs
 Post-operative infections
 Septicemia in the immunocompromised

78. BIOCHEMICAL IDENTIFICATION
 Lactose – Negative
 PDA – Positive
 Urease – Positive
 Ornithine – Positive
 Indole - Positive

79. PROVIDENCIA SPECIES

80. CLINICAL SIGNIFICANCE
 Nosocomial infections
 Wound infections
 Skin infections
 Septicemia

81. BIOCHEMICAL IDENTIFICATION
 Lactose – Negative
 Indole – Positive
 Citrate – Positive
 PDA – Positive
 MR – Positive
 VP- Negative

82. EDWARDSIELLA TARDA

83. CLINICAL SIGNIFICANCE
 Rare causes of GI infections
 Hepatic abscesses
 Meningitis
 Wound infections

84. BIOCHEMICAL IDENTIFICATION
Non-lactose fermenter
Gas production from glucose
IMVI:C (++--)
 Indole = Positive
 MR = Positive
 VP = Negative
 Citrate = Negative
H 2 S – positive
Lysine – Positive
Ornithine – Positive

85. CITROBACTER FREUNDII

86. CLINICAL SIGNIFICANCE
 UTIs
 Respiratory infections
 Septicemia
 Wound infections

87. BIOCHEMICAL IDENTIFICATION
 Lactose – Positive
 Gas production from glucose
 Lysine – Negative
 Urea – Positive (70%)
 Citrate = Positive
 H 2 S - Positive

88. Salmonella
ENTERIC PATHOGENS Shigella
Yersinia

89. SALMONELLA SPECIES

90. CLINICAL SIGNIFICANCE
 Salmonella typhi
 Not human normal flora
 Humans are the only reservoirs
 Ingestion of contaminated food products
 Fecal-oral transmission
 Typhoid fever

91. BIOCHEMICAL IDENTIFICATION
 Lactose = Negative
 H 2 S = Positive
 Gas production from glucose
 Lysine = Positive
 Urease, Indole & Citrate = Negative

92. SHIGELLA SPECIES

93. CLINICAL SIGNIFICANCE
 Shegellosis or bacterial dysentery
 Transmitted by ingesting contaminated food or water
 Fever
 Abdominal cramping and pain
 Scant stools with blood, mucous and pus
 Few organisms are capable of causing disease

94. BIOCHEMICAL IDENTIFICATION
 Non-motile
 Lactose = negative
 Anaerogenic
 MR = Positive
 Ornithine = Positive

95. YERSINIA SPECIES

96. CLINICAL SIGNIFICANCE
 Yersinia entercolitica
 Entercolitis
 Mimics acute appendicitis
 Yersinia pestis
 Bubonic plague
 Pneumonic plague

97. COLONY MORPHOLOGY

98. BIOCHEMICAL IDENTIFICATION
 Lactose – negative
 Motile at 22°C but not at 37°C

99. SUMMARY TABLES

100. ID THE BUG!

101. ID THE BUG - 1

102. ID THE BUG - 1 - ANSWER
IMViC = ++-- E. coli

103. ID THE BUG - 2

104. ID THE BUG - 2 - ANSWER
IMViC = --++ K. pneumoniae

105. WHAT’S NEXT?
 Review Mahon, Chapter 19
 Complete AUMoodle Review