ENTEROBACTERIACEACE

by: Dr. WILSON R. DELOS REYES JR.

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 FAMILY About t hi s t e mp l a t e
OF ENTEROBACTERIACEAE

Genus:
Budvicia Leclercia Obesumbacterium Trabulsiella
Buttiauxella Leminorella Pragia Xenorhabdus
Cedecea Morganella Pantoea Yersinia
Citrobacter Photorhabdus Yokenella
Edwardsiella Proteus
Enterobacter Providencia
Escherichia Rahnella
Ewingella Salmonella
Hafnia Serratia
Klebsiella Shigella
Kluyvera Tatumella

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 GENERAL CHARACTERISTICS

◉ Common term: Enterobacteria

◉ Facultatively anaerobic, non-sporeforming, Gram negative bacilli
◉ All members are m otile at 35C with peritrichous flagella, except:
Klebsiella, shigella and Yersinia
◉ All are non-encapsulated except for Klebsiella and Enterobacter
◉ All members are glucose fermenter and reduce nitrate to nitrite
◉ Most of them are present in the intestinal tract as commensal
microbiota except for Plesiomonas, Salmonella, Shigella, and Yersinia
◉ Some organisms Serratia may grow at 1C to 5C.

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 GENERAL CHARACTERISTICS

◉ Microscopy: Straight Gram negative rods or

coccobacilli with rounded ends.
◉ Culture on BAP: Colonies appear large, smooth and
gray except for Klebsiella and Enterobacter with
mucoid colonies (capsule) and are non-haemolytic
except for some strain of Escherichia coli
◉ Biochemical tests:
CORRECTED 
◉ Catalase Test: Positive
◉ Oxidase Test: Negative; except for Plesiomonas
shigelloides.

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 ANTIGEN-DETERMINANTS FOR IDENTIFICATION

◉ Somatic O Antigen: heat-stable; located in the cell

wall; used for E. coli and Shigella serotyping
◉ Flagellar H Antigen: heat-labile; found in the
flagellum; used for Salmonella serotyping
◉ Capsular K Antigen: heat-labile polysaccharide; found
as K1 antigen of E.coli and Vi antigen of S. enterica
subsp. enterica serotype Typhi

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 NOTE TO REMEMBER

◉ E. coli, K. pneumoniae and K. oxytoca: ESBL-producing

enterobacteria
◉ E. coli, P. mirabilis and K. pneumonia are isolated from
urinary tract and can cause bacteremia.
◉ Citrobacter, Enterobacter and Serratia are antibiotic
resistant genera.
◉ Shigella, Salmonella, E. coli and Yersinia are associated
with diarrhea.
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 LABORATORY DIAGNOSIS

• Gram stain • Triple Sugar Iron Agar Test • Phenylalanine deaminase

• Sulfide Indole Motility Test • Urea hydrolysisTest
• Culture • Citrate utilization Test
• Gelatin liquefaction Test
• Biochemical Test • Lysine Iron AgarTest
• Decarboxylase Test • Malonate Tes
• Molecular Test • Ortho-nitrophenyl--Galactopyranoside
• Methyl red/Voges-Proskauer (MRVP) Test
• Nitrate reductionTest

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 Gram Stain
• Straight gram-negative rods or coccobacilli with
rounded ends
• Direct smears of stool specimen are not routinely
performed
• Nice to know:
• Wayson Stain: used to observe the bipolar
bodies of Yersinia pestis
VIAS
V – Crystal Violet
I – Iodine
A – acetone
S - Safranine
 GRAM STAIN

◉ Straight Gram-negative rods or coccobacilli with

rounded ends.
◉ Direct smears of stool specimen are not routinely
performed
◉ Nice to know:
○ Wayson stain: used to observe the bipolar bodies
of Yersinia pestis

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 LACTOSE FERMENTATION

RAPID LACTOSE LATE LACTOSE NON-LACTOSE

FERMENTER FERMENTER FERMENTER
• Enterobacter • Citrobacter freundii • Citrobacter koseri
• Escherichia coli • Serratia • Proteus
• Klebsiella • Hafnia • Providencia
• Shigella sonnei • Morganella
• Salmonella arizoniae • Shigella
• Yersinia enterocolitica • Salmonella
• Yersinia
• Edwardsiella tarda

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 CULTURE

◉ Culture Media: BAP, CAP, MAC, HEA, XLD, CIN, SSA, EMB,
Bismuth sulphite Agar (BSA), Selenite F and GN Broth.
◉ Transport Media: Amies, Cary-Blair and Stuart transport media
◉ Colony morphology: BAP and CAP – Large, gray and smooth
◉ Fecal pathogen: Generally non-lactosefermenter
◉ Salmonella: produce colonies with black centers in media with
H2S as indicator: HEA, B S A and XLD.
◉ Optimal temperature: 35C to 37C; except:
○ Serratia and Yersinia (1C to 5C)
○ E. coli which can also grow12at 45C to 50C
 CULTURE

Ma cCon ke y - S or bi t o l Agar (MAC-SOR/SMAC)

◉ Used to differentiate E.coli

o157:H7 (sorbitol negative)
from other strains of E. coli
(sorbitol positive)
◉ pH indicator: Neutral Red
◉ Result: E. coli o157:H7
exhibits clear or colorless
colonies while other
strains are PINK 13
 CULTURE

HEKTOEN ENTERIC AGAR (HEA)

◉ Content: Bile salt,bromthymol

blue dye, salicin, lactose and
sucrose
◉ Bile salt and dye: inhibit the
gram-negative bacilli in GITand
promote the isolation of
Salmonella and Shigella.
◉ pH indicator: Bromthymol blue
◉ H2S indicator: Ferric ammonium
citrate
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 HEA RESULTS

◉ Lactose Fermenter: Yellow

color
○ Except: Citrobacter freundii (with
black center)
◉ Non-Lactose Fermenter:
Blue-green color
○ Except: Proteus and Salmonella
(green with black center
◉ Non-Enteric pathogens:
Orange to pinkish-orange
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 CULTURE

XYLOSE-LYSINE DESOXYCHOLATE (XLD) AGAR

◉ Useful in isolation of Salmonella

and Shigella species from heavily
contaminated specimen such as
stool.
◉ Contain lactose, sucrose, xylose,
lysine and sodium desoxycholate
◉ pH indicator: Phenol red
◉ H2S indicator: Sodium thiosulfate and
ferric ammonium sulphate
Shigella Pink to red colonies _
Salmonella Pink to red with black centers colonies
Other Yellow to red colonies
 CULTURE

EOSIN-METHYLENE BLUE (EMB) or LEVINE’s MEDIUM

◉ It contains eosin Y, methylene

blue, lactose and sucrose.
◉ pH indicator: Eosin and methylene
blue
◉ Result:
Lactose Colonies of E. coli exhibit a greenish
Fermenter metallic sheen while those of the other
LF’s exhibit purple color
Non-Lactose Colonies are colorless
Fermenter

Other Yellow to red colonies

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 CULTURE

SALMONELLA-SHIGELLA AGAR (SSA)

◉ It differentiate Salmonella and

Shigella from other enteric bacteria.
◉ Bile salts and Brilliant green dye:
Inhibits gram positive bacteria and
som e lactose fermenter that are
found in the stool specimens.
◉ Carbohydrates: Lactose
◉ pH indicator: Neutral red
◉ H2S indicator: Sodium thiosulfate and
ferric ammonium citrate
Salmonella Pink or colorless with black center
Shigella
Pink or colorless without black center
 SEROTYPING (AGGLUTINATION TEST)

◉ Identifies strains (serovars or ◉ 2. E. coli serotyping

serotypes) of microorganisms that
differ in their antigenic composition.
◉ Commonly tested organisms:
Salmonella, Shigella and E. coli
O157:H7
◉ Medium: BAP with 5% sheep’s blood.
◉ 1. Salmonella and Shigella serotyping
○ Salmonella serotyping: Heat-
stable somatic O antigen and
Heat-labile flagellar H antigen
○ Shigella serotyping: based on
the somatic O antigen
○ So rbitol-negative E. coli can
be serotyped to identify
whether som atic O antigen
and flagellar H antigen
◉ E. coli O55, O111, and O127:
infantile diarrhea
◉ Result: (+)Agglutination
Salmonella serotype Typhi: Producesheat-
labile capsular polysaccharide Vi antigen
and carries the D serogroup
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 LABORATORY DIAGNOSIS

• Biochemical Test • Triple Sugar Iron Agar Test • Phenylalanine deaminase

• Sulfide Indole Motility Test • Urea hydrolysisTest
• Citrate utilization Test
• Gelatin liquefaction Test
• Lysine Iron AgarTest
• Decarboxylase Test • Malonate Test
• Ortho-nitrophenyl--Galactopyranoside
• Methyl red/Voges-Proskauer (MRVP) Test
• Nitrate reductionTest

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 BIOCHEMICAL TEST Glucose, lactose, sucrose

FERMENTATION OF SUGAR – TRIPLE SUGAR IRON AGAR

◉ Fermentation of Sugar is usually

detected by acid production
◉ Gram negative rod utilizes g lucose
and lactose or sucrose or not. Slant  Sucrose and Lactose
◉ Identify bacteria producing g as and TSIA = original (orange or red)
H2S Acid
◉ Two necessary enzyme for an = Yellow (Lactose or Sucrose
enterobacterium to take up lactose fermenter)
○ -galactoside permease:
Acid = yellow
Facilitates the entry of the lactose If there is black, it is
molecule positive with H2s

-galactosidase: hydrolyzes lactose

into glucose and galactose If there are bubbles, it isIf yellow, glucose fermenter (GF)
gas producer Butt - glucose
 BIOCHEMICAL TEST

FERMENTATION OF SUGAR – TRIPLE SUGAR IRON AGAR

Characteristics of TSIA: Interpretation of TSIA:

◉ Red color at pH 7.4 ◉ Bacterial specie that are incapable of
◉ Ratio of sugars: 10:10:1 (lactose, fermenting glucose cannot utilize
sucrose and glucose) lactose
◉ pH indicator: Phenol red ◉ Lactose fermenter posses both B-
galactoside permease and B-galactoside
◉ H2S indicator: Ferrous sulphate and
Sodium thiosulfate ◉ Non-lactose fermenters (NLF) do not
◉ Should not read beyond 24 of posses B-galactoside permease and B-
incubation. galactosidase
◉ True enteric pathogens only ferment ◉ Late Lactose fermenter: only B-
glucose. galactosidase
◉ Glucose and lactose fermenters are
mostly opportunistic enterics
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 BIOCHEMICAL TEST

FERMENTATION OF SUGAR – TRIPLE SUGAR IRON AGAR

TSIA REACTION: C. LACTOSE, SUCROSE AND GLUCOSE

A. NO FERMENTATION OF SUGARS:
◉ Result: Alkaline slant/Alkaline butt
(K/K)
◉ Not Enterobacteriaceae
B. NO LACTOSE AND SUCROSE
FERMENTATION (Glucose fermenter)
◉ Result: Alkaline slant/Acid butt (K/A)
◉ Glucose concentration depleted
◉ After 18-24hrs of incubation, organism
will utilize PEPTONE in the slant. Lead
to alkaline products (RED)
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FERMENTATION
◉ Result: Acid slant/Acid butt
◉ Enterobacteriaceae attack glucose first
and then dissacharide
D. HYDROGEN SULFIDE (H2S) production
◉ H2S indicator: Sod ium thiosulfate and
ferrous sulphate
◉ Result: (+) Formation of black ppt.
E. GAS PRODUCTION
◉ Result: Formation of bubble (CO2 and H2),
splitting of media in butt or complete
displacement at the bottom.
 BIOCHEMICAL TEST

FERMENTATION OF SUGAR – TRIPLE SUGAR IRON AGAR

TSIA REACTION: C. LACTOSE, SUCROSE AND GLUCOSE

FERMENTATION
A. NO FERMENTATION OF SUGARS:
◉ Result: Alkaline slant/Alkaline butt ◉ Result: Acid slant/Acid butt
Gas Producer:
(K/K) ◉ Enterobacteriaceae
Escherichiaattack
coli glucose first
and thenEnterobacter
dissacharideaerogenes
◉ Not Enterobacteriaceae
H2S Producer: D. HYDROGENEnterobacter cloacae
SULFIDE (H2S) production
B. NO LACTOSE AND
Proteus mirabilis SUCROSE Klebsiella pneumonia
FERMENTATION Proteus vulgarisfermenter)
(Glucose ◉ H2S indicator: Sodoxytoca
Klebsiella ium thiosulfate and
Citrobacter freundii ferrous sulphate
Serratia marcescens
◉ Result: Alkaline slant/Acid butt (K/A)
Salmonella serotype Typhi Proteus mirabilis
◉ Glucose Edwardsiella
concentration depleted
tarda
◉ Result: (+) Formation of black ppt.
Morganella morganii
E. GAS PRODUCTION
Citrobacter freundii
◉ After 18-24hrs of incubation, organism
Salmonella serotype Typhi
will utilize PEPTONE in the slant. Lead ◉ Result: Formation of bubble (CO2 and H2),
Edwardsiella tarda
to alkaline products (RED) splitting of media in butt or complete
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displacement at the bottom.
 BIOCHEMICAL TEST

SULFIDE INDOLE MOTILITY (SIM) TEST

◉ (+) Sulfide: Black color formation in the butt

◉ (+) Indole: Pink to “wine-colored” ringformation after
the addition of Kovac’s reagent
◉ (+) Motility: Movement away from stab line that
produces a hazy appearance.
◉ Note: Kovac’s reagent should be added into the SIM
medium after 18 to 24 hours of incubation.

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 BIOCHEMICAL TEST

CITRATE UTILIZATION TEST

◉ Determine the ability of an organism to utilize

○ sodium citrate: carbon source
○ Inorganic ammonium salts: nitrogen source
◉ (+) Result:
◉ pH indicator:
◉ Note: If citrate is used as carbon source
○ Green medium 

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 BIOCHEMICAL TEST

CITRATE UTILIZATION TEST

◉ Determine the ability of an organism to utilize

○ sodium citrate: carbon source
○ Inorganic ammonium salts: Citrate positive
Nitrogen source
Enterobacter aerogenes
Enterobacter cloacae
Klebsiella pneumonia
◉ (+) Result: Blue colored citrateagar Klebsiella oxytoca
Serratia marcescens
slant Proteus mirabilis (v)
Providencia rettgeri
◉ pH indicator: Bromthymol blue Providencia stuartii
Citrobacter freundii
◉ Note: If citrate is used as Citrobacter koseri
Hafnia alvei
carbon source (alkaline)
○ Green medium  Blue medium (alkaline)
 Proteus
BIOCHEMICAL TEST
Providencia
Morganella
LYSINE IRON AGAR (LIA) TEST

Interpretation:
◉ Organisms secretes lysine decarboxylase 
cadaverine (purple color). Lysine  cadavene
◉ If not produce decarboxylase  butt remains acidic
(Yellow color)
◉ Deamination of Lysine  Form burgundy red color on
slant
◉ Deamination does not occur  Slant remain purple
◉ Glucose fermenter  30
butt becomes acidic (yellow)
LIA REACTION LIA REACTION INTERPRETATION RELATED BACTERIA

Alkaline slant K (-) Lysine deamination Salmonella

Alkaline butt K (+) Lysine decarboxylation

Alkaline slant K (-) Lysine deamination Shigella

Citrobacter
Acid butt A (-) Lysine decarboxylation

Red slant R (+) Lysine deamination Proteus

Providencia
Acid butt A (-) Lysine decarboxylation Morganella

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 BIOCHEMICAL TEST Arginine, Lysine, Ornithine

DECARBOXYLASE TEST (MOELLER’S METHOD)

◉ Measures enzymatic ability of an organism to decarboxylate (or

hydrolyze) an amino acid to form an amine (putrescine or cadaverine).
◉ Moeller decarboxylase media: Glucose, arginine, lysine and ornithine
◉ pH indicator: Bromcresol purple or phenol red
◉ Promotes anaerobiosis: Mineral oil
◉ Incubation condition: Four days
Positive Result Alkaline product (Purple or Red) Enterobacter sp.
Negative Result Glucose fermenter (Yellow Klebsiella sp.
color)
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 Lysine decarboxylase
Lysine  cadaverine

Arginine decarboxylase
Arginine  citrulline

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 BIOCHEMICAL TEST

ORTHO-NITROPHENYL--GALACTOPYRANOSIDE (ONPG)

◉ Identifies slow or late

lactose fermenters
◉ -galactosidase acts on
the ONPG: cleaves it
into galactose and
ortho-nitrophenol
◉ Positive result: Yellow
color within 20 minutes Serratia, Hafnia, Citrobacter tyeundii
◉ If NLF: compound S. arizonae, S.sonnei, Y. enterocolitica
remain colorless 32
 BIOCHEMICAL TEST

METHYL RED/VOGES-PROSKAUER TEST

◉ Determine the ability of an

organism to produce and
maintain stable acid end
products (pyruvic acid) from
glucose fermentation
◉ Culture media in M R test:
MRVP broth/ Peptone
glucose broth
◉ M R reagent: Methyl red
◉ VP reagents: 40% KOH and 1%
a-naphthol
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BIOCHEMICAL TEST

METHYL RED/VOGES-PROSKAUER TEST

Example:
MR/VP: Escherichia coli (+/-)

MR/VP: Klebsiella – Enterobacter –

Serratia – Hafnia Group (-/+)

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 BIOCHEMICAL TEST

NITRATE REDUCTIONTEST

◉ Can reducenitrate to nitrite

◉ Detects nitrate reductase
production of enterobacteria.
“+”
◉ Reagents: (change
○ Sulfanillic acid in color
○ -naphthylamine from “+”
reagents) (no change in
◉ (+) Result: red, water-soluble color from zinc;
positive from
◉ Zinc dust: confirm negative zinc dust)
reaction

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 BIOCHEMICAL TEST

NITRATE REDUCTIONTEST

◉ Can reducenitrate to nitrite

◉ Detects nitrate reductase
production of enterobacteria.
◉ Reagents:
○ Sulfanilic acid
○ -naphthylamine
◉ (+) Result: Red, water-soluble
azo dye
◉ Zinc dust: confirm negative
reaction

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 BIOCHEMICAL TEST

PHENYLALANINE DEAMINASE (PAD) TEST

◉ Used for differentiation of

Proteus, Morganella and
Providencia, only PAD-
positive genera. Proteus
◉ Deaminate phenylalanine to Providencia
phenyl pyruvic acid Morganella

◉ Confirmatory reagent:
10% ferric chloride
◉ (+) Result: green-colored slant_
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 BIOCHEMICAL TEST

UREA HYDROLYSIS TEST (CHRISTENSEN’S METHOD)

◉ Ability of an organism to
produce the urea
hydrolysing enzyme urease.
◉ Reagent: Urea disk dissolved
in 1-mL distilled water.
◉ End product: Ammonium
carbonate( alkaline product)
Positive Slant (orange  Magenta)
Negative Yellow
Rapid urase Proteus and Morganella
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 BIOCHEMICAL TEST

GELATIN LIQUEFACTION TEST

◉ Determine the production in

the bacteria of gelatinase
(protease) which hydrolyze
and liquefies thegelatin.
◉ Culture media:Nutrient
gelatine tube agar
◉ (+) Result: Liquefied gel

Positive Serratia and Proteus

Negative Escherichia coli

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 BIOCHEMICAL TEST

MALONATE TEST

◉ Determines if capable of
utilizing sodium malonate as
it’s sole carbon source.
◉ (+)Result: Exhibits a blue
color at pH 7.6
◉ Enterobacter and
Salmonella are positive

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 LABORATORY DIAGNOSIS

• Molecular Testing

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 MOLECULAR TESTING

◉ Enhance recovery and

identification of enteric
bacteria.
◉ Reclassifies genus
Plesiomonas as enteric GI
pathogens.
◉ Methods:
○ 16S ribosomal RNA
sequencing
○ D N A - DNA hybirdization
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 MOLECULAR TESTING

◉ Enhance recovery and

identification of enteric
bacteria.
◉ Reclassifies genus
Plesiomonas as enteric GI
pathogens.
◉ Methods:
○ 16S ribosomal RNA
sequencing
○ D N A - DNA hybirdization
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SUMMARY

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 Th a n k s !

ANY QUESTIONS?

You can find me at:

📌 Viber: 09666145106
📖
wrdelosreyes@fatima.edu.ph

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