This document outlines the objectives and content to be covered regarding the Enterobacteriaceae family of bacteria. It will describe the general characteristics, taxonomy, biochemical profiles and reactions, growth properties, and clinical significance of various genera within the family including Escherichia, Klebsiella, Proteus, Salmonella, Shigella and others. Specific tests such as lactose fermentation, TSI, urease, citrate, and motility will be discussed. Important opportunistic pathogens will be highlighted and methods for differentiating clinically relevant species will be presented.

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