Gram Negative

Bacilli
Enterobacteriaceae
 Enterobacteriaceae
Enterobacteriaceae is a family of gram-negative rods inhabiting the
intestinal tract of humans and animals.
The Enterobacteriaceae grow rapidly under aerobic or anaerobic conditions and are metabolically
active
• They are by far the most common cause of urinary tract infections (UTIs), and a limited number of
species are also important etiologic agents of diarrhea. Entry into the bloodstream may cause Gram-
negative
endotoxin shock, a dreaded and often fatal complication
The Enterobacteriaceae are among the largest bacteria, measuring 2 to 4 μm in length with parallel
sides and rounded ends
• Enterobacteriaceae grow readily on simple media, often with only a single carbon energy source.
Growth is rapid under both aerobic and anaerobic conditions, producing 2 to 5 mm colonies on agar
media and diffuse turbidity in broth after 12 to 18 hours of incubation. All Enterobacteriaceae ferment
glucose, reduce nitrates to nitrites, and are oxidase negative.
Components of the cell
wall and surface, which
are antigenic

The outer membrane

lipopolysaccharide (LPS) is called the
O antigen
Cell surface polysaccharides may
form a well defined
capsule or an amorphous slime layer
and are termed the K antigen
Motile strains have protein
peritrichous flagella, which extend
well beyond the cell wall and are
called the H antigen
Diagnosis
• Specimen collection: stool, anal swabs, and other clinical material
depending upon the localization of the infection
• included urine, blood, pus, spinal fluid, sputum, or other material, as indicated by the
localization of the disease process.
Microscopy:
• The Enterobacteriaceae are among the largest bacteria, measuring 2 to 4 μm in length with parallel
sides and rounded ends. Forms range from large coccobacilli to elongated ,filamentous rods. The
organisms do not form spores

• Culture
 culture
According to their ability to ferment Lactose enterobacteriace are
classified into:
• Lactose Fermenters: E. coli, Klebsiella, Citrobacter &
Enterobacter
• Non Lactose: Fermenters: Salmonella, Shigella & Proteus
Lactose fermentation on MacConkey medium
Bile salts inhibit all m.o Except Enterobacteriaceae
lactose fermenters (coliform)  produce acid
 change (Neutral red)color of indicator to pink
- non-lactose fermenters ( Salmonella, Shigella)  yellow colonies
- Selective & differential media

Uses: isolation of enterobacteriaceae from clinical specimen

- differentiate lactose fermentors from non- lactose fermentors
Eosin Methylene Blue (EMB)

- Eosin + M.B. inhibit all m.o Xpt.

enterobacteriaceae
-typical coliforms produce high conc.
of acid enough to
ppt. Eosin  green metallic sheen
- atypical  dark colonies – no sheen
Selective, differential
- isolation of enterobacteriaceae
- differentiation bet.
typical coliforms (as E. coli) &
atypical coliforms (as Klebsiella
Biochemical Tests used in Identification of
Enterobacteriaceae
• Sugar Fermentation Test
• Triple Sugar Iron Test
• IMViC Test.
• Urease
Sugar Fermentation test
• Enteric bacteria can ferment different types of sugars (glucose, lactose,
Maltose, sucrose) producing either acid only or acid and gas
• Thus sugar fermentation tests are useful for primary identification of strains by
determining the following.
• Type of the sugar.
• End product (acid or acid & gas )
• Conditions ( aerobic or anaerobic )
• Media used for sugar utilization test usually consist of:
• Peptone
• Single fermentable sugar
• Acid / Base Indicator
• Durham Tube
Sugar -ve acid/ -ve gas
No color change
+ve acid/-ve gas
Carbohydrate is
+ve acid /+ve
gas

Fermentation test The organism

couldn't ferment the
fermented without
production of gas
sugar and utillized the e.g. Salmonella
Carbohydrate
fermented with
acid and gas (+)
peptone as carbon Shigella
source instead.
Andrad’s reagent Acid / Base Indic.: ( red in e.g. Pseudomonas
(e.g.Esch.coli,
acid, Yellow in Alkaline pH) Klebsiella)
Phenol red (yellow in acid, red in Alkaline
pH)
 Reactions in triple sugar iron agar

TSI : a) acid slant & acid but

b) alkaline slant & acid butt
c) alkaline slant + H2S

D) E. coli: acid slope, acid butt, + ve gas

C)Shigella: alkaline slope, acid butt
E) Salmonella: Alkaline slope, acid butt ,
+ve H2S, + ve gas
Uses: Differentiate bet. Enterobacteriaceae
Nitrate reduction

• This test detects the presence

of enzyme nitrate reductase
which causes the reduction of
nitrate to nitrite
• Almost all Enterobacteriaceae
members reduce nitrate
• Nitrate reduction can be
detected by an appropriate
colorimetric reagent
• Uses:Test for the presence of Nitrate
reductase enzyme in Enterobacteriace
IMViC Test

IMViC test is a group of four

biochemical test collectively used
for primary identification of
enteric bacteria.
• Indole
• Methyl Red.
• Vogeus proskuer.
• Citrate Utillization.

IMViC ++-- IMViC --++

Indole Test • This test demonstrates the ability of
certain bacteria to decompose the
amino acid Tryptophan to Indole
• This indole when combined with a
compound called para dimethyl
amino benzaldehyde
(Kovacs reagent) gives a pink
colored compound
• Result
• Pink colour Indole positive bacteria
– Escherichia coli
• No colour Indole negative bacteria
– Klebsiella spp.
Methyl Red

• This test is used to detect the

production of large amount of acid
during the fermentation of glucose
• The acidity can be determined by
adding a few drops of methyl red
solution, as indicator
• Result
• Bright red Positive (e.g. Escherichia
coli)
• Yellow Negative (e.g. Klebsiella
spp)
Voges-Proskauer

• Some bacteria ferment carbohydrates

with the production of acetyl methyl
carbinol
• This breakdown product when reacts
with Alpha-Naphthol in alcohol in the
presence of alkali gives a cherry red
color
• Result
• Pink or cherry red colour VP Positive
(e.g. Klebsiella)
• Yellow colour VP negative (e.g.
Escherichia coli)
Citrate Utillization

• Some bacteria can utilise simple

organic salts like sodium citrate
as the sole source of carbon and
energy source for growth
• During such utilization CO2 is
liberated which makes the
medium alkaline
• Simmon’s citrate agar may be
used
Urease Test

• Some bacteria, particularly

those growing naturally in an
environment exposed to urine,
may decompose urea by
enzyme urease and liberate
ammonia
• Urea Ammonia + CO2 +
Water
• The production of the enzyme
urease is detected by growing
the organisms in the presence
of urea and testing for alkali
(NH3) Phenol red pH indicator
ESCHERICHIA
COLI
 E.coli

E COLI EXTRAINTESTINAL INFECTIONS

1- URINARY TRACT INFECTION uropathogenic E coli (UPEC).
Minor trauma admits E coli to the bladder (Type 1 pili adhere to periurethral and bladder cells, P pili prominent in
pyelonephritis)
• signs include urinary frequency, dysuria, hematuria, and pyuria. Flank pain is associated with upper
tract
2- OTHER EXTRAINTESTINAL INFECTIONS
Meningitis
E coli is one of the most common causes of neonatal meningitis
3- E COLI INTESTINAL INFECTIONS (E coli-Associated
Diarrheal Diseases)
• causes of moderate to severe diarrhea among children
• Diarrhea causing E coli are classified according to their virulence
properties as enterotoxigenic (ETEC)(“traveler’s diarrhea” produce a heat-labile
exotoxin (LT)), enteropathogenic (EPEC) (is an important cause of diarrhea in
infants), enter invasive (EIEC)(disease very similar to shigellosis),
enterohemorrhagic (EHEC)(hemorrhagic colitis), or enteroaggregative
(EAEC)(causes acute and chronic diarrhea).
Morphology

• Shape: gram Negative Rods

• Arrangement: Single
• Non spore forming, non-Capsulated & motile
Diagnosis
Specimen collection: stool, anal swabs, and other clinical material
depending upon the localization of the infection
• Culture Characteristics
• N.Agar: Circular, convex and small smooth colonies.
• MacConkeys medium: Rose pink colonies indicating lactose fermentations
• Eosin Methylene blue (E.M.B): Metallic Sheen colonies
 KLEBSIELLA
• The most distinctive bacteriologic features of the genus Klebsiella are the
absence of motility and the presence of a polysaccharide capsule gives
colonies a glistening, mucoid character and forms the basis of a serotyping
system
• Morphology
• Shape: gram Negative Rods
• Arrangement: Single
• Capsulated, Non spore forming & non-motile
• Culture Characteristics
• N.Agar: Mucoid, convex and smooth colonies.
• MacConkeys medium: Rose pink colonies indicating lactose fermentation
• Biochemical Reaction:
• IMViC : --++
• TSI: Acid but / Acid Slant with gas production
• Klebsiella pneumoniae, the most common species, is able to cause
classic lobar pneumonia,
 Salmonella

Non-Lactose Fermenters
• Morphology
• Shape: gram Negative Rods
• Arrangement: Single
• Non spore forming, non-Capsulated & motile
• Biochemical Reactions
• Lactose & sucrose non-fermenters
• Ferment Glucose, Maltose, Mannose & mannite
• Salmonella typhi production of acid only.
• Salmonella paratyphi produce acid & gas
• Reaction on TSI: Alkaline slant, acid Buttom with H2S production.
• Culture Characteristics
• Nutrient Media
• N. Agar: Circular, convex and small smooth colonies.
• Selective Differential Media
• MacConkeys medium: Pale colonies indicating non-lactose
fermentation
• Eosin Methylene blue (E.M.B): colorless colonies
• Bismuth sulphite agar (Wilson andBlair medium): Salmonella
gives black colonies with metallic sheen due to H2S
• Taylor’s Xylose Lysine Deoxycholate: Red colonies with black center.
• Enrichment Media:
• Tetrathionate, Selenite F Broth
Deoxycholate Citrate Agar (Leifson’s): Simillar to
Macconkey’s with black center
SALMONELLOSIS

The clinical patterns of salmonellosis can be divided into gastroenteritis, bacteremia with or without focal
extraintestinal infection, enteric fever, and the asymptomatic carrier state
• SALMONELLA GASTROENTERITIS (S enterica = gastroenteritis) Diarrhea, vomiting, and cramps
are common
ENTERIC (TYPHOID) FEVER
• Enteric fever is a multiorgan Salmonella infection characterized by prolonged fever, sustained
bacteremia, and profound involvement of the mesenteric lymph nodes, liver, and spleen
The manifestations of typhoid
Typhoid fever is a strictly human disease; chronic carriers of S Typhi are the primary reservoir(There is no
animal model for the strictly human S Typhi), Slowly increasing fever lasts for weeks
Transmission is by the fecal–oral route
• Three serotypes called Paratyphi (A, B, C) have features similar to S Typhi, including the production of
an enteric fever syndrome
Diagnosis
Culture of Salmonella from
the blood or stool is the
primary diagnostic method
Stool and blood culture are
routine
Identification of a salmonella by
slide agglutination (Widal)
This test can be used in
identification and
differentiation of different
Salmonella species using
Specific H- and O- antisera.
SHIGELLA
Shigella is also spread by food or water contaminated by human waste
products
Non-Lactose Fermenters
• Morphology
• Shape: gram Negative Rods
• Arrangement: Single
• Non spore forming, non-Capsulated & non-motile
• Biochemical Reactions
• All strains are Lactose & sucrose non-fermenters
• Non- Mannitol Fermenters:
• Shigella Dysenteriae which ferment glucose only.
• Mannitol Fermenters:
• Sh. Flexneri, Sh. Boydii, Sh. Sonii ferment glucose and mannitol
• Growth on TSI: Acid bottom /alkaline slant without H2S production.
• Deoxycholate Agar (Leifson’s): Translucent colonies without black center
differ from those of salmonella.
 DIAGNOSIS

Shigella organisms cause an acute inflammatory colitis

and bloody diarrhea, which in the most characteristic
state presents as a dysentery syndrome
Mortality significant with S dysenteriae type 1
Watery diarrhea followed by fever, bloody mucoid
stools, and cramping
Mortality significant with S dysenteriae type 1
All Shigella species are readily isolated using selective
media, stool culture protocol in all clinical laboratories
• Slide agglutination tests using O group specific
antisera (A, B, C, D) confirm both the species and the
Shigella genus.
 Proteus

Non-Lactose Fermenters
• Morphology
• Shape: gram Negative Rods
• Arrangement: Single
• Non spore forming, non -Capsulated
• Motile with peritrichous flagella.
• Biochemical Reactions
• Lactose & sucrose non-fermenters
• Pvulgaris, P. mirabilis +ve H2S
• Urease Positive
Swarming of Proteus on N.
Agar

Culture Media:
• Grow on ordinary media producing concentric growth
zone ( swarming)
• On XLD show black colonies due to H2S production
 II-Pseudomonas
• Morphology
Other gram-negative • Shape: gram Negative Rods
• Arrangement: Single
bacilli • Non spore forming, non-
Capsulated & motile
• Produce different exopigments
with different colors
• Biochemical Reactions
• Can't ferment sugar but oxidize
glucose to form acids (strict
aerobes)
• Oxidase + Ve
• Citrate + Ve
• Culture Characteristics
• Bluish green coloration on
nutrient agar as result of pigment
production.
• Heamolysies on blood agar.
 Diagnosis
• Pili (fimbriae) extend from the cell surface and promote
attachment to host epithelial cells
• Specimens from skin lesions, pus, urine, blood, spinal fluid,
sputum, and other material should be obtained as indicated by
the type of infection
• Gram-negative rods are often seen in smears. There are no
specific morphologic characteristics that differentiate
pseudomonads in specimens from enteric or other gram-
negative rods
• Culture
• Specimens are plated on blood agar
• Cetrimide agar selective media used to isolation of
Pseudomonas From clinical specimen
Biochemical reaction Oxidase (Test for
cytochrome oxidase
enzyme)
Precaution !
Use glass rod or
Platinum loop To
avoid false +ve
 Vibrio cholerae
• transmission in water and the development of
sanitary water systems.
• V cholerae is a comma-shaped, curved rods It is
actively motile by means of a polar flagellum. On
prolonged cultivation, vibrios may become straight
rods that resemble the gram-negative enteric
bacteria
• V cholerae regularly ferments sucrose and mannose
• Many vibrios share a single heat-labile flagellar H
antigen. Antibodies to the H antigen are probably not
involved in the protection of susceptible hosts
• V cholerae produce a heat-labile enterotoxin
Diagnosis

• culture
• V.cholerae grows well on thiosulfate-citrate-bile-
sucrose (TCBS) agar, on which it produces
yellow colonies that are readily visible against the
dark-green background of the agar
• Vibrios are oxidase-positive, which
differentiates them from enteric gram-negative
bacteria
Rapid detection

• V cholerae organisms are further identified by slide

agglutination tests using anti-O group 1 or group 139
antisera and by biochemical reaction patterns