ENTEROBACTERIACEAE

General Characteristics

 gram-negative bacilli
 aerobic and facultatively anaerobic
 non–spore-forming
 non-motile or peritrichously flagellated
 oxidase-negative,
 produce acid fermentatively from glucose
 reduce nitrates to nitrites
 Pathogenesis
URINARY TRACT, THOSE FREQUENTLY ISOLATED ARE
E. coli
Proteus mirabilis,
 Klebsiella pneumoniae.
PNEUMONIA
 Klebsiella pneumoniae.
BACTEREMIAS
E. coli
Proteus mirabilis,
 Klebsiella pneumoniae.
 Pathogenesis

HOSPITAL ACQUIRED INFECTIONS (antibiotic resistant genera)

Citrobacter, Enterobacter, and Serratia
DIARRHEA
 Shigella spp.,
Salmonella spp.,
E. coli (enterohemorrhagic [Shiga toxin producing], enterotoxigenic,
enteroinvasive, enteropathogenic, enteroadherent),
Yersinia spp.
 Pathogenesis
 Donovanosis
Klebsiella granulomatis
Virulent Factors

 Endotoxins

 Lipopolysaccharides

 K1
antigen
E. coli causing neonatal meningitis
 Capsule

 Vi
antigen
Salmonella serotype typhi,
Media
Biochemical tests
 OXIDASE TEST

Principle: Cytochrome oxidase participates in

electron transport and in nitrate metabolism
Reagent: 1% tetramethyl-p-phenylenediamine
dihydrochloride
Positive: dark blue
Caution: Do not use iron containing wire, do
not interpret results after 10 seconds
 NITRATE REDUCTION

PRINCIPLE:
 The
organisms produce nitrate reductase, which converts the nitrate (NO3) to nitrite
(NO2).
Nitrite + SULFANILIC ACID = diazonium salt
diazonium salt + alphanaphthylamine = red, water-soluble azo dye (+)
* If no color change occurs, the organism did not reduce nitrate or reduced it
further to NH3, NO, or N2O2.
No color change + zinc = red (NEGATIVE RESULT)
No color change + zince = no color change (POSITIVE RESULT)
 TRIPLE SUGAR IRON AGAR TEST

Principle:
Determine whether a gram negative rod utilizes glucose, and lactose or
sucrose fermentatively and forms hydrogen sulfide
 TRIPLE SUGAR IRON AGAR TEST

Principle:
 Composition:
10 parts lactose
10 parts sucrose
1 part glucose and peptone
Phenol red – indicator of acidification
Ferric ammonium citrate– indicator of Hydrogen sulfide
 Incubation
period
18-24 hours
 Result
Example Reaction on TSI
Result Slant Butt
H2S
color color
Non fermenter
e.g. Alk/Alk/- Negative Red Red
Pseudomonas (No action on sugars)

LNF A/Alk/- Negative

e.g. Shigella (Glucose fermented Red Yellow
without H2S)

LNF Positive
e.g. Salmonella & A/Alk/+ black in
Proteus (Glucose fermented butt Red Yellow
with H2S)

LF
e.g. E. coli, A/A/- Negative
Klebsiella, (three sugars are Yellow Yellow
Enterobacter fermented)
 Indole

Principle: tryptophanase
Reagent: 1% paradimethyaminocinnamaldehyde
Positive: blue within 20 seconds
Caution: Inoculum should not be selected from MacConkey agar
Indole Production (tube)
 Kovac’s reagent (dimethylamine-benzaldehyde and hydrochloride
red color
Enterobacteriaceae
 Ehrlich’sreagent
same chemicals as the Kovac preparation, + absolute ethyl alcohol
more sensitive for detecting small amounts of indole.
also be used to differentiate organisms under anaerobic conditions.
Positive: Pink- to wine-colored ring after addition of appropriate reagent
Indole Production (tube)

KOVAC’S REAGENT EHRLICH’S REAGENT

Indole
 Methyl Red/Voges Proskauer (MR/VP)

PRINCIPLE

METHYL RED - used to determine the ability of an organism to produce and

maintain stable acid end products from glucose fermentation
Voges Proskauer - determine the ability of some organisms to produce neutral
end products (e.g., 2,3-butanediol or acetoin) from glucose fermentation.
 Methyl Red/Voges Proskauer (MR/VP)

 REAGENT

MRVP Broth
Voges Proskauer - alpha-naphthol, potassium hydroxide (KOH)
 RESULT

Positive – red color

Negative – yellow color
 Methyl Red/Voges Proskauer (MR/VP)

Methyl Red: left – and right + VP: left + and right –

 Citrate Utilization Test

PRINCIPLE

using sodium citrate as the sole carbon source and inorganic ammonium
salts as the sole nitrogen source.
use the citrate and convert ammonium phosphate to ammonia and ammonium
hydroxide, creating an alkaline pH.
The pH change turns the bromthymol blue indicator from green to blue.
 Citrate Utilization Test

 REAGENT

Simmon’s citrate agar

 RESULTS

Positive – blue color

Negative – green color
Citrate Utilization Test

Left positive and right negative.

 H2S Production in SIM
 Principle

determine the ability to reduce sulfur into H2S.

 Reagents :
SIM media contains the sulfur containing amino acid, cysteine, sodium
thiosulfate, & peptonized iron or ferrous sulfate.
 RESULTS

Positive – black
 Urease Test

Principle: detects the enzyme urease

Reagent: 1% paradimethyaminocinnamaldehyde
Positive: pink
Caution : Alkaline reactions may appear after prolonged incubation and
may be the result of peptone or other protein utilization raising the pH.
 Motility Test

 PRINCIPLE

used to determine whether an enteric organism is motile.

 REAGENT

Semi-solid media
 RESULTS

Positive - diffuse zone of growth extending out from the line of

inoculation
 Motility Test

From left to right:

+ – +
 LYSINE IRON AGAR (LIA)
 PRINCIPLE

differentiate gram-negative bacilli based on decarboxylation or deamination

of lysine and the formation of hydrogen sulfide
glucose is fermented, the butt of the medium becomes acidic (yellow)
Cadaverine neutralizes the organic acids formed by glucose fermentation, and
the butt of the medium reverts to the alkaline state (purple).
If oxidative deamination of lysine occurs, a compound is formed that, in the
presence of ferric ammonium citrate and a coenzyme, flavin mononucleotide,
forms a burgundy color on the slant.
 LYSINE IRON AGAR (LIA)
 RESULTS

Alkaline slant/alkaline butt (K/K)—lysine decarboxylation and no fermentation

of glucose
Alkaline slant/acid butt (K/A)—glucose fermentation
Red slant/acid butt (R/A)—lysine deamination and glucose fermentation
 LYSINE IRON AGAR (LIA)
 RESULTS
 PHENYLALANINE DEAMINASE

 PRINCIPLE

Microorganisms that produce phenylalanine deaminase remove the amine

(NH2) from phenylalanine. The reaction results in the production of ammonia
(NH3) and phenylpyruvic acid.
The phenylpyruvic acid is detected by adding a few drops of 10% ferric
chloride; a green colored complex is formed
 RESULTS

Positive – green color

PHENYLALANINE DEAMINASE
o-Nitrophenyl-β-D-Galactopyranoside (ONPG) Test

 usedto determine the ability of an organism to produce β-

galactosidase
 distinguisheslate lactose fermenters from non–lactose fermenters
of Enterobacteriaceae.
 Positive: Yellow
o-Nitrophenyl-β-D-Galactopyranoside (ONPG) Test
 4-Methylumbelliferyl-β-D-Glucuronide (MUG) Test

 usedto presumptively identify various genera of

Enterobacteriaceae and verotoxin-producing
Escherichia coli.
 Verotoxin
producing strains of E. coli do not
produce MUG
 Positive: Electric blue fluorescence
BIOCHEMICAL
IDENTIFICATION
 Oxidase Test
Negative Positive

Enterobacteriaceae Pseudomonas
MacConkey’s agar
& TSI  O/F test: O+/F-
 Nitrate test: +ve further
Pink colonies on MacConkey colorless colonies on MacConkey reduction to N2
& acidic butt and slant on TSI & acidic butt alkaline slant onTSI
 Growth on cetrimide agar:
Lactose fermenter Lactose non-fermenter Pale colonies with green
pigmentation
IMViC test No H2S production H2S production
& EMB (no blacking in TSI) (blacking in TSI)

IMViC IMViC Urease production

++ - - - - ++ Shigella
& black colonies +ve -ve
with metalic Motility
shines on EMB SS agar
Not motile Motile colorless colonies with black centers
E.coli Proteus
Salmonella
Escherichia coli

BIOCHEMICAL TESTS RESULT

IMViC ++--

H2S -

PAD -

TSI K/K
 Escherichia coli

 Enterotoxig enic Escherichia coli (ETEC)

traveler’s diarrhea
profuse watery diarrhea with no blood nor leukocytes and
abdominal cramping
The stool looks like rice water just like cholera!
 Enteroinvasive Escherichia coli (EIEC)
diarrhea is bloody with white blood cells.
 Like shigellosis!
 Escherichia coli

 Enterohemorrhagic Escherichia coli (EHEC):

Shiga toxin-producing E. coli (STEC)
Verocytotoxin-producingE. coli (VTEC)
This pathotype is the one most commonly heard about in the news in
association with foodborne outbreaks.
Hemorrhagic colitis.
Hemolytic uremic syndrome (HUS) - Escherichia coli 0157:H7
 Escherichia coli

 Enteropathogenic Escherichia coli

Non-invasive
Produces no toxin
Nosocomial
Watery diarrhea with mucus but no blood
Escherichia coli
 Kleibsiella
K. pneumoniae
Friedlander’s bacillus
 Lactose positive mucoid colonies
 Enterobacter

 E.
sakazaki
Yellow pigmentation that intensifies at 25°C
 E.
cloacae
Most predominant isolate
 LDC ODC ADH IMViC

K. pneumoniae + - - --++

K. oxytoca + - - +v++

E. aerogenes + + - --++

--++
E. cloacae - + +

P. agglomerans - - - vvvv
Citrobacter

 UTI and sepsis

 Resembles Salmonella but ONPG + and LDC -
 C.
freundii – H2S +
IMVC: v+-v
 C.
diversus (C. koseri) – H2S -
IMVC: ++-+
 Serratia
 Serratia
marcescens may produce a red pigment
PRODIGIOSIN
 DNAse, Gelatinase and Lipase positive

IMVC
S. marcescens -v++
S. liquefaciens -+++
 Salmonella
 produce H2S
 colonies with black centers on XLD, HE, and bismuth sulfite agars.
***selenite-F or gram-negative (GN) broth - useful for detecting low
numbers of Salmonella spp. and Shigella spp. in stool.
 IMVC: -+-+
 Motile
 Salmonella

 Serotypingis based on immunologic reactivity of the heat-stable somatic

“O” antigens, which are predominantly lipopolysaccharide in content,
and the heat-labile flagellar “H” antigens.
 Salmonella serotypes typhimurium and enteritidis are the most common
 Salmonella serotype typhi also produces a heat-labile capsular
polysaccharide Vi antigen
Salmonella

 Serogroups
Serogroup A - S. paratyphi A
Serogroup B - S. paratyphi B
Serogroup C - S. cholerasuis
Serogroup D - S. typhi
 Shigella

 Agent of bacillary dysentery

Group
Present designation Catalase ONPG Mannitol IMVC
Type
S. dysenteriae A - - - v+--
v+--
S. flexneri B + - +
v+--
S. boydii C + - +
S. sonnei D + + + -+--
 Proteus

 Certainstrains strains share specific polysaccharides with some rickketsia

and are agglutinated by sera from patients with ricketssial disease.
 Source of Antigen for Weil-Felix Reaction (use to diagnose Rickketsial
infection)
P. vulgaris – OX-2 and OX-19
P. mirabilis – OX-K (Kingsburg)
Swarming motility (swarming on BAP)
 Proteus

IMVC
P. mirabilis -+vv
P. vulgaris ++-v
P. penneri -+--
Proteus-Providencia-Morganella group

Non-lactose fermenter
Motile
Methyl red positive
R/A in LIA
Phenylalanine deaminase +
Lysine deaminase +
Proteus-Providencia-Morganella group

IMVC
P. rettgeri ++-+
P. stuartii ++-+
M. morganii ++--
Edwardsiella tarda

 Differentiate
from E. coli
NON-Lactose Fermenter
Hydrogen sulfide positive
IMVIC : ++--
 Yersinia

 Motile at room temperature

 Non-motile at 37°C
 Y.
pestis
Agent of plague (black death)
Transmitted by the rat flea (Xenopis cheopsis)
Broth cultures : stalactite pattern
Inclusion: bipolar bodies
“Safety pin” appearance on Wayson’s stain
Yersinia

 Yersinia
enterocolitica
Cefsulodin irgasan- novobiocin
(CIN) agar incubated at room
temperature is selective and
differential
bull’s-eye colonies with red
centers and transparent
borders.
IMVC: v+--
Hafnia alvei

 Formerly Enterobacter alvei

 Late lactose fermenter
 Citrate negative
 IMVC : -vv-
Plesiomonas shigelloides

 facultatively anaerobic
 oxidase- and catalase-positive
 glucose-fermenting,

 gram-negative rod
 most closely related to the genus Proteus
 only oxidase-positive member
 Plesiomonas shigelloides

 minimum growth temperature of 8°

 Implicatedas a cause of gastroenteritis, especially following the
ingestion of uncooked shellfish.
 non–lactose fermenter.
 indole positive; reduces nitrates to nitrites
 methyl red positive; and ferments glucose, maltose, and trehalose