Chapter 9

ENTEROBACTERIACEAE & 19
 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.
 TAXONOMY

Family
Enterobacteriaceae

Genera
Escherichia Serratia
Shigella Proteus
Edwardsiella Morganella
Citrobacter Providencia
Klebsiella Yersinia
Enterobacter Salmonella
 GENERAL CHARACTERISTICS

Found in nature
Plants
Soil
Water

Intestines of humans

Most are normal enteric flora

 BIOCHEMICAL CHARACTERISTICS

Ferment glucose
Oxidase negative
Reduce nitrate to nitrite
Oxidase Test Nitrate Reduction

Glucose Fermentation
 MICROSCOPIC CHARACTERISTICS

Gram negative bacilli

Most are motile
Non-spore forming
 GROWTH REQUIREMENTS

Facultative anaerobes
Incubation length
18-24 hours
Incubation temperature
35-37C
Incubation atmosphere
Air
 MACROSCOPIC CHARACTERISTICS

Grow well on BAP

Grow on enteric media
MAC
HE
XLD
EMB
 THE SCOOP ON POOP!

Enterobacteriaceae are routinely isolated from stool cultures

ID work-up
Only performed on those that are pathogens
ENTERIC MEDIA
 BLOOD AGAR PLATE - 1

All Enterics grow on

BAP
Allows for
observation of colony
true morphology
BLOOD AGAR PLATE - 2
 MACCONKEY (MAC) - 1

Selective
Gram negative

Differential
Lactose fermentation
Fermenter
PINK
Non-fermenter
Clear
MACCONKEY (MAC) - 2
XYLOSE-LYSINE-DESOXYCHOLATE (XLD) - 1

Selective
Bile Salts
Differential
Lactose fermentation
YELLOW
H2S
BLACK
 XYLOSE-LYSINE-DESOXYCHOLATE (XLD) - 2

Lactose Non-Fermenter; H2S Producer Lactose Fermenter

 EOSIN-METHYLENE BLUE (EMB) - 1

Selective
Gram negative
Differential
Lactose fermentation
BLUE-BLACK
EOSIN-METHYLENE BLUE (EMB) - 2
 HEKTOEN ENTERIC (HE) - 1

Selective
Gram negative
Differential
Lactose fermentation
YELLOW/ORANGE
SALMON (Pink)
H2S
BLACK
HEKTOEN ENTERIC (HE)
BIOCHEMICAL REACTIONS
 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
TRIPLE SUGAR IRON AGAR (TSI) - 1

Media composition
Sugars (Glucose, Lactose, Sucrose)
Peptone - nutrient and energy
pH indicator
H 2 S indicator
 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
TSI REACTIONS - 1
TSI REACTIONS - 2
TSI REACTIONS - 3
TSI REACTIONS - 4
TSI REACTIONS - 5
TRIPLE SUGAR IRON AGAR (TSI)
Test Your Knowledge
 O-F TESTS

See power point presentation for chapter 21.

 INDOLE

Tryptophanase + tryptophan pyurvic acid + ammonia + indole

indole + 1% paradimethylaminocinnamaldehyde color change (pink color)
 UREA

Urease + urea (broth or agar) ammonia + water + CO2

Ammonia produces a pH change in the media = Pink
 UREASE

Tube 1: Control

Tube 2: Proteus vulgaris

= Positive

Tube 3: E. coli
= Negative
CITRATE

+ = +
 DNASE

Positive

Escherichia coli (top):

negative-no clearing

Serratia marcescens (bottom):

positive-clearing
DECARBOXYLASE TESTS - ORNITHINE

Control Positive Negative Control

DECARBOXYLASE TESTS ARGININE &
LYSINE
 METHYL RED

Glucose Pyruvic acid Mixed acid fermentation (pH 4.4)

Acid end-products detected with methyl red indicator
 VOGES-PROSKAUER

Glucose Pyruvic acid Acetoin Diacetyl

Diacetyl + KOH + -naphthol Red complex
 ONPG

Lactose fermenter have the -

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
MOTILITY

Motility is defined as
any growth away
from the stab
1 = Non-motile
2 = Motile
3 = Motile
MOTILITY/INDOLE/ORNITHINE (MIO)

Indole Reaction

Motility Reaction

Ornithine Reaction
 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.
 NITRATE

Add Zinc dust

= =
+ ? = =
?

Nitrate Nitrite
Nitrite + sulfanilic acid + N,N-Dimethyl-1-naphthlamine red
 NITRATE

Positive

Positive

Negative
 GELATIN HYDROLYSIS

Escherichia coli: negative

(gelatin is solid)
Pseudomonas aeruginosa: positive (gelatin has been liquefied)
ANALYTICAL PROFILE INDEX (API)
API 20-E
Negative Tests

Positive Tests
OPPORTUNISTIC
ENTERICS
ESCHERICHIA COLI
 CLINICAL SIGNIFICANCE

Most common human pathogen

UTI
Septicemia
Meningitis
Pneumoniae
Endocarditis
 COLONY MORPHOLOGY

BAP
MACHE
XLD
EMB
 MICROSCOPIC EVALUATION

Gram negative
bacilli
Bipolar staining
 BIOCHEMICAL IDENTIFICATION

Lactose fermenter
Gas from Glucose
IMV:C (++--)
Indole Positive
MR Positive
VP Negative
Citrate Negative

ONPG Positive
Lysine - Positive
E. COLI Serotypes
 ENTEROHEMMORAGIC E. COLI
(EHEC)
E. coli 0157:H7
Hemorrhagic colitis
Hemolytic Uremic Syndrome (HUS)
Low platelets
Hemolytic anemia
Kidney failure
 ENTEROHEMMORAGIC E. COLI
(EHEC)
MacConkey with
Sorbitol

Negative = clear/colorless

Positive = PINK
KLEBSIELLA PNEUMONIAE
 CLINICAL SIGNIFICANCE

Most common isolate

Lower respiratory infections in hospitalized patients
Pneumonia
Septicemia
Meningitis
UTI
 COLONY MORPHOLOGY

Very mucoid (wet) looking

Due to a polysaccharide capsule
 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)
ENTEROBACTER
 CLINICAL SIGNIFICANCE

Opportunistic pathogens
E. aerogenes
E. cloacae bacteremia and wound infections
Meningitis
Endocarditis
Urinary tract infection
Lower respiratory tract infections
 BIOCHEMICAL IDENTIFICATION

Lactose = Positive
Motile
IMV:C (--++)
Indole = Negative
MR = Negative
VP = Positive
Citrate = Positive
Ornithine = Positive
SERRATIA SPECIES
 CLINICAL SIGNIFICANCE

Nosocomial
UTI
Respiratory infections
Septicemia
Endocarditis
 COLONY MORPHOLOGY

Red pigmentation
S. rubidea
S. marcescens
Smells like peeled
potatoes
S. odorifera
 BIOCHEMICAL IDENTIFICATION

Lactose = Negative
IMV:C (-+++)
Indole = Negative
MR = Positive
VP = Positive
Citrate = Positive
PROTEUS SPECIES
 CLINICAL SIGNIFICANCE

Nosocomial infections
UTIs
Wound infections
Septicemia
Pneumonia
 COLONY MORPHOLOGY

Swarm on BAP
Smells like burnt chocolate
 BIOCHEMICAL IDENTIFICATION

Lactose negative
PDA - Positive
Urease Positive
H 2 S Positive (most)
 SPECIES DIFFERENTIATION

P. mirabilis
Indole = Negative
Ornithine = Positive

P. vulgaris
Indole = Positive
Ornithine = Negative
MORGANELLA SPECIES
 CLINICAL SIGNIFICANCE

Nosocomial infections
UTIs
Post-operative infections
Septicemia in the immunocompromised
 BIOCHEMICAL IDENTIFICATION

Lactose Negative
PDA Positive
Urease Positive
Ornithine Positive
Indole - Positive
PROVIDENCIA SPECIES
 CLINICAL SIGNIFICANCE

Nosocomial infections
Wound infections
Skin infections
Septicemia
 BIOCHEMICAL IDENTIFICATION

Lactose Negative
Indole Positive
Citrate Positive
PDA Positive
MR Positive
VP- Negative
EDWARDSIELLA TARDA
 CLINICAL SIGNIFICANCE

Rare causes of GI infections

Hepatic abscesses
Meningitis
Wound infections
 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
CITROBACTER FREUNDII
 CLINICAL SIGNIFICANCE

UTIs
Respiratory infections
Septicemia
Wound infections
 BIOCHEMICAL IDENTIFICATION

Lactose Positive
Gas production from glucose
Lysine Negative
Urea Positive (70%)
Citrate = Positive
H 2 S - Positive
 Salmonella
ENTERIC PATHOGENS Shigella
Yersinia
SALMONELLA SPECIES
 CLINICAL SIGNIFICANCE

Salmonella typhi
Not human normal flora
Humans are the only reservoirs
Ingestion of contaminated food products
Fecal-oral transmission
Typhoid fever
 BIOCHEMICAL IDENTIFICATION

Lactose = Negative
H 2 S = Positive
Gas production from glucose
Lysine = Positive
Urease, Indole & Citrate = Negative
SHIGELLA SPECIES
 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
 BIOCHEMICAL IDENTIFICATION

Non-motile
Lactose = negative
Anaerogenic
MR = Positive
Ornithine = Positive
YERSINIA SPECIES
 CLINICAL SIGNIFICANCE

Yersinia entercolitica
Entercolitis
Mimics acute appendicitis

Yersinia pestis
Bubonic plague
Pneumonic plague
COLONY MORPHOLOGY
 BIOCHEMICAL IDENTIFICATION

Lactose negative
Motile at 22C but not at 37C
SUMMARY TABLES
ID THE BUG!
ID THE BUG - 1
 ID THE BUG - 1 - ANSWER

IMViC = ++-- E. coli

ID THE BUG - 2
 ID THE BUG - 2 - ANSWER

IMViC = --++ K. pneumoniae

 WHATS NEXT?

Review Mahon, Chapter 19

Complete AUMoodle Review