ENTEROBACTERIACEAE

JOMARI B. CADDAUAN
 ENTEROBACTERIACEAE
• Gram negative rods
• Non-spore forming
• Facultative anaerobes
• Ferment glucose
• Reduce nitrate to nitrite
___________________________________
Except Erwinia and Pantoea agglomerans
 ENTEROBACTERIACEAE
• Motile with peritrichous flagella
Except Klebsiella and Shigella
_______________________________
• Catalase positive
____________________________
Except Shigella dysenteriae
• Oxidase negative
_______________________________
Except Plesiomonas shigelloides
 Antigenic Structure
• K antigen
CAPSULAR ANTIGEN
– ________________ Some Salmonellae have K
– ________________
HEAT LABILE antigens, referred to as Vi
• O antigen
– ________________
SOMATIC ANTIGEN Antibodies to O antigens
– ________________
HEAT STABLE are predominantly IgM
• H antigen
– ________________
FLAGELLAR ANTIGEN H antigens agglutinate
– ________________
DENATURED BY HEAT with anti-H antibodies,
AND ALCOHOL mainly IgG
Antigenic Structure
 Laboratory Identification
1. Growth on Media
A. MAC
B. EMB
C. HEA
D. SSA
 Laboratory Identification
RAPID LACTOSE LATE LACTOSE NON-LACTOSE
FERMENTERS FERMENTERS FERMENTERS
• Escherichia • Citrobacter • Proteus
• Klebsiella • Hafnia • Providencia
• Enterobacter • Yersinia • Morganella
• Salmonella arizonae • Salmonella
• Shigella sonnei • Edwardsiella
• Shigella
• Erwinia

EKE CHYSS PPMoSESE

 Laboratory Identification
A. MACCONKEY (MAC) AGAR

MAC SELECTION AND DIFFERENTIATION OF

GRAM NEGATIVE ENTERIC BACTERIA
INHIBITORS CRYSTAL VIOLET AND BILE SALTS
CHO LACTOSE
pH INDICATOR NEUTRAL RED
LACTOSE FERMENTERS PINK COLONIES
NON-LACTOSE FERMENTER COLORLESS COLONIES
 Laboratory Identification
A. MACCONKEY (MAC) AGAR
 Laboratory Identification
A. EOSIN-METHYLENE BLUE (EMB) AGAR
EMB SELECTION AND DIFFERENTIATION OF
GRAM NEGATIVE ENTERIC BACTERIA
CHO LACTOSE
LACTOSE FERMENTERS PINK TO PURPLE COLONIES
E. coli – pink to purple with green metallic sheen
Klebsiella – pink mucoid colonies “FISH EYE
Enterobacter – pink colonies with dark center COLONIES”
NON-LACTOSE FERMENTER COLORLESS COLONIES
 Laboratory Identification
B. EOSIN-METHYLENE BLUE (EMB) AGAR
 Laboratory Identification
C. HEKTOEN-ENTERIC (HEA) AGAR
HEA SELECTION AND DIFFERENTIATION OF GRAM NEGATIVE
ENTERIC BACTERIA
INHIBITORS BILE SALTS
CHO LACTOSE, SUCROSE, SALICIN
pH INDICATOR BROMTHYMOL BLUE
H2S INDICATOR FERRIC AMMONIUM CITRATE
LACTOSE FERMENTER, H2S - YELLOW COLONIES WITHOUT BLACK CENTER
LACTOSE FERMENTER, H2S + YELLOW COLONIES WITH BLACK CENTER
NON-LACTOSE FERMENTER, H2S - GREEN COLONIES WITHOUT BLACK CENTER
NON-LACTOSE FERMENTER, H2S + GREEN COLONIES WITH BLACK CENTER
 Laboratory Identification
C. HEKTOEN-ENTERIC (HEA) AGAR
 Laboratory Identification
D. SALMONELLA-SHIGELLA (SS) AGAR
SSA SELECTIVE AND DIFFERENTIAL FOR
SALMONELLA AND SHIGELLA
CHO LACTOSE
pH INDICATOR NEUTRAL RED
H2S INDICATOR FERRIC CITRATE
SALMONELLA COLORLESS COLONIES WITH BLACK
CENTER
SHIGELLA COLORLESS COLONIES WITHOUT
BLACK CENTER
 Laboratory Identification
2. TRIPLE SUGAR IRON (TSI) AGAR
COMPOSITION

PROTEIN SOURCES Beet extract, peptone, yeast extract,

proteose peptone
CARBOHYDRATES 10 parts LACTOSE, 10 parts SUCROSE, 1 part GLUCOSE

pH INDICATOR PHENOL RED

H2S INDICATOR FERROUS SULFATE

 Laboratory Identification
2. TRIPLE SUGAR IRON (TSI) AGAR
TSI REACTIONS CHO FERMENTED TYPICAL ORGANISMS

A/A G+ H2S- LACTOSE + SUCROSE + Escherichia

Klebsiella
GLUCOSE + Enterobacter

K/A G+ H2S+ LACTOSE - SUCROSE - Citrobacter

Salmonella
GLUCOSE + Proteus

K/A H2S- LACTOSE - SUCROSE -

Shigella
GLUCOSE +

K/K H2S- LACTOSE - SUCROSE -

Pseudomonas
GLUCOSE - Alcaligenes
Laboratory Identification
 Laboratory Identification
3. LYSINE IRON AGAR (LIA)
COMPOSITION
SMALL AMOUNT OF PROTEIN SOURCES

CARBOHYDRATES GLUCOSE
AMINO ACID LYSINE
pH INDICATOR BROMCRESOL PURPLE
H2S INDICATOR FERRIC AMMONIUM CITRATE
 Laboratory Identification
3. LYSINE IRON AGAR (LIA)
LIA REACTIONS TYPICAL ORGANISMS

K/K H2S+ LDA -

LDC +
Salmonella
K/A H2S- LDA -

LDC - Shigella
R/A H2S- LDA + Proteus
Providencia
LDC - Morganella
Laboratory Identification
 Laboratory Identification
4. INDOLE

• Detects ____________
Trypyophanase
• Medium _______________
SIM, Trypticase Broth
• Indicator ____________________
KOVAC’s/Ehrlich reagent

p-dimethylaminobenzaldehyde

• Positive result _________________

Formation of RED RING
 Laboratory Identification
5. MRVP Test
• Medium ___________
MRVP Broth

a. METHYL RED
 Organism that produce acid will overcome
the neutralizing effect of the buffer
 Positive result _____
RED
 Laboratory Identification
5. MRVP Test
• Medium ___________
MRVP Broth

b. VOGES PROSKAUER
Acetoin
 Detects __________
KOH, a-naphtol
 Reagents _______________
RED
 Positive Result _____
 Laboratory Identification
6. CITRATE UTILIZATION

• Medium _______________
Simmon Citrate Agar
• Indicator _________________
Bromthymol Blue
• Positive result ____
BLUE
 ESCHERICIA
I M V C
E. coli
KLEBSIELLA
I M V C
K. pneumoniae
K. oxytoca
K. ozaenae
ENTEROBACTER
I M V C
E. aerogenes
E. cloacae
E. agglomerans
 SERRATIA
I M V C
S. marcescens
S. liquefaciens
HAFNIA
I M V C
H. alvei
SALMONELLA
I M V C
Most Serotypes
SHIGELLA
I M V C
ABC
D
 CITROBACTER
I M V C
C. freundii
C. diversus/koseri
PROTEUS
I M V C
P. mirabilis
P. vulgaris
P. penneri
PROVIDENICIA
I M V C
P. rettgeri
P. stuartii
 MORGANELLA
I M V C
M. morganii subsp.
morganii
EDWARSIELLA
I M V C
E. tarda
YERSINIA
I M V C
Y. enterocolitica
Y. frederisenii
Y. intermedia
 Laboratory Identification

7. Malonate Utilization

• Positive ____
BLUE
• Negative __________
Green or Yellow
 Laboratory Identification
8. UREASE TEST

Christensen’s Urease Agar / Stuart’s

• Medium _____________________________
Urease Agar

• Indicator ___________
Phenol Red
• Positive result ____
RED
 Laboratory Identification

8. UREASE TEST
Proteus
Providencia
RAPID UREASE PRODUCERS Morganella
PPM
Citrobacter
SLOW UREASE PRODUCERS Klebsiella
Enterobacter
CKEYS Yersinia
Serratia
 Laboratory Identification

9. ONPG
o-nitrophenyl-beta-D-galactopyranoside

• Rapid Test to detect _____________

B-galactosidase
• Positive _______
Yellow
 Laboratory Identification

10.Decarboxylase
 Positive _________________
Purple to Yellow to Purple
11.PAD PPM
Green Slant and Fluid
 Positive _________________
Proteus
12.Gelatin liquefaction Providencia
 Positive _____________
Liquefaction Morganella
 Escherichia
IMViC TSI
 Escherichia

• UTI - 90%
• Sepsis
• Meningitis
• Diarrheal diseases
 Escherichia
1. Enterotoxigenic E. coli (ETEC)
• Produces heat labile and heat stable
enterotoxins
• LTs are closely related in structure and
function to ____________
Cholera toxin
• Montezuma’s revenge
PROFUSE
Traveler’s diarrhea WATERY
STOOL
Childhood diarrhea
 Escherichia
2. Enteroinvasive E. coli (EIEC)

• Invades the intestinal epithelium causing

_____________________
Shigella-like toxin
• Dysentery
• _________________________
STOOL WITH RBC,
NEUTROPHIL AND MUCUS
 Escherichia
3. Enteropathogenic E. coli (EPEC)

• Produces NO TOXIN
Non-invasive, ____________
• Nosocomial, ______________
Seen in newborn and infants
• ___________________________
WATERY STOOL WITH MUCUS
BUT NO BLOOD
 Escherichia
4. Enterohemorrhagic E. coli (EHEC) or
___________________
Verotoxin E. coli (VTEC)
Shiga-like toxin
• Produces verotoxin
Named for its cytotoxic effect on Vero cells
• ________
HUS - most severe manifestation of EHEC
• Most frequently associated with certain
serotypes such as E. coli 0157:H7
 Escherichia

• SMAC
– MacConkey Agar in which _____________
SORBITOL has
been substituted foe lactose
E. coli 0157:H7
– _________________
• Only strain that is sorbitol negative
• Colorless colonies
 Escherichia
5. Enteroaggregative E. coli (EAEC)

• Probably involves binding with pili, ST-like,

and hemolysin-like toxins; actual pathogenic
mechanism not known.
• WATERY STOOL
_____________________
 Klebsiella
IMViC TSI
 Klebsiella

• Exhibit mucoid growth

• Large polysaccharide capsule

1. _________________
K. pneumoniae
– ____________________
FRIEDLANDER’S BACILLUS
– Encapsulated
– Appears as mucoid colonies that tend to string
 Klebsiella

2. ______________
K. ozaenae
– Purulent sinus infection

3. ________________
K. rhinoscleromatis
– Granuloma of the nose and oropharynx
 Enterbacter
IMViC TSI
 Enterobacter

• Opportunistic infections: UTI and wound

infections
• Most predominant isolate is
_______________
E. cloacae
• Produces yellow that intensifies at 25 C
_______________
E. sakazakii
 LDC ODC ADH
K. pneumoniae
K. oxytoca
E. aerogenes
E. cloacae
P. agglomerans
Serratia
 Serratia

• S. marcescens and S. rubidea produce a

red pigment ____________
Prodigiosin
• _____________________
S. odorifera produces a rancid
potato-like odor
• Common opportunistic pathogen in
hospitalized patients
Salmonella
 Salmonella
Water, milk and dairy products,
• Sources of infection: shellfish, dried or frozen eggs,
meat and meat products
• Cause enteritis, systemic infection and enteric
fever __________
Typhoid Fever
• Cause enteric fever:
– ______________________
S. paratyphi A (serogroup A)
– ______________________
S. paratyphi B (serogroup B)
– ______________________
S. cholerasuis (serogroup C)
– ______________________
S. typhi (serogroup D)
– ______________________
S. typhi (most common cause)
 Salmonella

• Bacteremia
– _______________
S. cholerasuis

• _____________
Enterocolitis/gastroenteritis
– Most common manifestation of salmonella
infection
S. typhimurium (most common)
– _________________
 Salmonella

• ___________
WIDAL TEST
– tube dilution agglutination test
O>1:160
– High or rising titer of ____________ suggests
that active infection is present
– High titer of ____________
H>1:160 suggests past
infection or immunization
– High titer of antibody to Vi antigen occurs in
some CARRIERS
Shigella
 GROUP TYPE CATALASE ONPG MANNITOL
S. dysenteriae

S. flexneri

S. boydii

S. sonnei
Citrobacter
 Citrobacter

• Causes UTI and sepsis

• Organisms resemble Salmonella
__________________________________
ONPG positive and LDC negative
Proteus
 Proteus

• Produce infections in humans only when

they leave the intestinal tract
• UTI, Bacteremia, Pneumonia, Nosocomial
Infections
• P. mirabilis and many strains of P. vulgaris
exhibit _____________
swarming
motility
 Proteus

• Certain strains share specific

polysaccharides with some rickettsia and
are agglutinated by sera from patients
with rickettsial diseases
________________________
P. vulgaris – OX-2 and OX-19
___________________
P. mirabilis – OX-K
Providencia
 Providencia

• Normal intestinal flora

• UTI and occasionally other infections and
are often resistant to antimicrobial
therapy
Edwarsiella
 Edwarsiella

• Isolated from the environment and many

cold blooded and warm blooded
animals including reptiles, freshwater and
aquarium fish, frogs and turtles
E. tarda
• _______________ - most human species
• To differentiate from E. coli
______________________
H2S+, non-LF
Yersinia
 Yersinia
Y. pestis
1. _________________
• Plague
• Xenopsylla cheopsis
Common vector: ______________
• Produced pandemics of black death with
millions of fatalities
• Broth cultures exhibit a characteristic
______________
Stalactite pattern
Bipolar bodies
 Inclusion ______________
 Stain __________________
Wayson’s Stain
 Yersinia

2. _________________
Y. enterocolitica
• Enterocolitis
• Bacteremia
• Cultured on ______________________
CIN
 ADDITIONAL NOTES

• ESBL – producing enterobacteria

E. coli, K. pneumoniae, K. oxytoca
• Antibiotic-resistant genera
Citrobacter, Enterobacter, Serratia
• Associated with diarrhea
Shigella, Salmonella, E. coli, Yersinia
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