ENTEROBACTERIACEAE

FAMILY
LECTURE ON
E.COLI, KLEBSIELLA,
PROTEUS
Dr Praveg Gupta MD
Dr Praveg Gupta MD 1
Dr Praveg Gupta MD 2
 ENTEROBACTERIACEAE
• A large Family of aerobic bacterial flora of intestine of
humans and other animals.
• Its members are nonsporting, non acid-fast, gram
negative bacilli.

• Capsule ±
• Motility ±

• General features – aerobic and facultatively anaerobic,

grow readily on ordinary media, ferment glucose, reduce
nitrates to nitrites and form catalase but not oxidase.

Dr Praveg Gupta MD 3
Catalase, glucose, nitrate +ve; oxidase -ve

Dr Praveg Gupta MD 4
• Wide biochemical and antigenic heterogeneity.

• Genetic mechanisms like conjugation and transduction in

these bacteria contribute to their infinite variety.

• Various classifications of Enterobacteriaceae have been

put forward.

• Two important classifications are 1. based on taxonomy

and 2. based on lactose fermentation.

Dr Praveg Gupta MD 5
 CLASSIFICATION BASED ON LACTOSE
FERMENTATION
1. Lactose fermenters
• Escherichia coli
• Klebsiella sp.

2. Late lactose fermenters

• Shigella sonnei
• Para colons etc

3. No lactose fermenters
• Salmonella
• Shigella etc.
Dr Praveg Gupta MD 6
Lactose fermenter v/s non fermenter

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 TAXONOMICAL CLASSIFICATION
ENTEROBACTERIACEAE

• Tribe I: Escherichia • Tribe III: Proteae

– Genus
– Genus
• Escherichia
• Proteus
• Edwardsville
• Morganella
• Citrobacter
• Providencia
• Salmonella
• Shigella
• Tribe IV: Erwinieae
– Genus
• Tribe II: Klebsiella
• Erwinia
– Genus
• Klebsiella
• Enterobacter
• Hafnia
• Serratia Dr Praveg Gupta MD 8
• Genus Escherichia named after Escherichia who was
the first to describe the colon bacillus under the name
Bacterium coli commune (1885).

 Species:
• E.coli,
• E.fergusonii,
• E.hermanii,
• E.vulneris,
• E.blattae etc

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ESCHERICHIA COLI

Dr Praveg Gupta MD 10
 MORPHOLOGY
• Gram negative bacilli

• 1-3 x 0.4-0.7 µm

• Single, pairs

• Motile by peritrichate flagella

• Found in some – capsules, fimbriae, immobility

• Non spore forming

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SHOWING GRAM NEGATIVE BACILLI
AND PERITRICHOUS FLAGELLA

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 CULTURE CHARACTERISTICS
• Aerobe and facultative anaerobe
• 10-40°C (37°C)

• S = smooth forms seen in fresh isolates, easily

emulsifiable in saline.

• R = rough forms seen in older cultures, with irregular dull

surface, often autoagglutinable in saline.

• S-R variation occurs as a result of repeated subcultures

and is associated with the loss of surface antigens and
usually of virulence.
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• Many pathogenic isolates have polysaccharide capsules.
• Some strains may occur in the mucoid form.

• Nutrient agar – colonies are large, thick, greyish white,

moist, smooth, opaque or partially translucent discs.

• Blood agar - Many strains esp. pathogenic ones are

hemolytic on blood agar.

• MacConkey medium - colonies are bright pink due to

lactose fermentation.

• Broth – general turbidity, heavy deposit.

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ECOLI ON NUTRIENT AGAR

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ECOLI ON BLOOD AGAR

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ECOLI IN BROTH

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E.COLI ON MACCONKEY AGAR

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 BIOCHEMICAL REACTIONS

• Sugar fermentation – glucose, lactose, manitol, maltose

and many other sugars fermented with acid and gas
production.

• Sucrose generally not fermented.

• IMViC ++--

• Gelatin -, H2S -, urease -.

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BIOCHEMICAL REACTIONS OF E.coli

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 ANTIGENIC STRUCTURE
• O = somatic antigen
• K = capsular antigen
• H = flagellar antigen

• So far, >170 types of O, 100 types of H and 75 types of K

have been identified.
• Antigenic pattern of an organism based on these antigens
is written as eg. O111:K58:H2, O54:K27:H41 etc.

• K antigen is the acidic polysaccharide antigen located in

the envelope or microcapsule (K for kapsel, german for
capsule).
• It encloses the O antigen and renders the strain
inagglutinable by the O antiserum.
• It may also contribute to virulence by inhibiting
phagocytosis.
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 VIRULENCE FACTORS

 Surface antigens: O and K

• O antigen – somatic lipopolysaccharide surface O

antigen has endotoxic activity, protects the bacteria from
phagocytosis and bactericidal effects of complement.

• K antigen – affords protection against phagocytosis and

antibacterial factors in normal serum.

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 Fimbriae – plasmid coded, found in small numbers and
mediate mannose resistant hem agglutinins, act as
virulence factors.

• Examples:

• CFA = colonization factor antigens in enter toxigenic

E.coli causing human diarrhea.

• P fimbriae which bind to uroepithelial cells and P blood

group substance on human erythrocytes, have a role in
urinary tract infection.
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 TOXINS:

 E.coli produce two kinds of exotoxins – haemolysins and

enterotoxins.

 Three distinct types of E.coli enterotoxins have been

identified –

• LT = heat labile toxin

• ST = heat stable toxin
• VT = serotoxin (also known as SLT = shiga like toxin).

Dr Praveg Gupta MD 24
 LT = HEAT LABILE TOXIN

• Resembles cholera toxin in its structure, antigenic

properties and mode of action.

• It is a complex of polypeptide subunits.

• Each unit of toxin has 1 subunit A and 5 subunits B.

• CT is about 100 times more potent than LT.

• LT is a powerful antigen and can be detected by

serological and biological tests.

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 LT

1 SUBUNIT A

5 SUBUNITS B
bind to GM1 ganglioside
Receptors on intestinal
epithelial cells

activation of A subunit into A1 and A2

A1 activates adenyl cyclase in the enterocyte to form

cAMP, leading to increased outflow of water and electrolytes
into the gut lumen, with consequent diarrhoea.
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 ST = HEAT STABLE TOXIN
• LMW polypeptide, poorly antigenic.
• Two types known – STA/ST1 and STB/ST2.
• ST genes are carried on plasmids which may also carry
other genes, such as for LT and drug resistance.

STA STB
STA acts by activation of cGMP in M/A not known.
the intestine.

Infant mouse test –STA acts very STB causes fluid accumulation in
rapidly and induces fluid young piglets but not in infant mice.
accumulation in the intestines of
infant mice within four hours of
intragastric administration.

Methanol soluble Not methanol soluble.

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 VT = VEROTOXIN = VEROCYTOTOXIN

• Named so because it was first detected by its cytotoxic

effect on Vero cells, a cell line derived from African
green monkey kidney cells.

• It is also known as SLT = shiga like toxin because it is

similar to the shigella dysenteriae type 1 toxin in its
physical, antigenic and biological properties.

• Has A and B subunits.

• VT genes appear to be phage encoded.
• VT1, VT2 identified.

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 CLINICAL INFECTIONS
• URINARY TRACT INFECTIONS
• DIARRHEA
• PYOGENIC INFECTIONS
• SEPTICAEMIA

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 URINARY TRACT INFECTIONS

• Community acquired: E.coli and other coliforms

account for the large majority of naturally acquired UTIs.

• Hospital acquired: Those acquired in the hospital,

following instrumentation, are more often caused by
other bacteria such as pseudomonas and proteus.

• The E.coli serotypes commonly responsible for UTI are

those normally found in the feces, O groups
1,2,4,6,7,etc.

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 Asymptomatic bacteriuria:

• Observed in some pregnant women, it means urinary

infection without any symptoms.
• If it progresses, it may lead to symptomatic infection later
in pregnancy, pyelonephritis and hypertension in the
pregnant woman, as well as to prematurity and perinatal
death of the fetus.

 Urinary tract infection can be:

• Ascending = via urethra
• Descending = haematogenous route

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 KASS’ CONCEPT OF SIGNIFICANT BACTERIURIA

• Normal urine is sterile, but during voiding may get

contaminated by genital commensals.
• Hence presence of bacteria in microscopy and culture of a
urine sample need not necessarily mean UTI by that
organism.
• To differentiate between actual pathogen and
contaminant, Kass etc. devised the following formula.

• Colony count in urine:

• <10000/ml = contaminant
• 10000 to 100000/ml = indecisive, repeat test.
• >100000/ml = significant bacteriuria.

• Exceptions: in patients on antibacterial or diuretic drugs

and with some bacteria like staph. Aureus, even low
counts may be significant.
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 URINE SAMPLE COLLECTION AND TRANSPORT

• Inform the patient about the procedure.

• Proper cleaning of area.
• Clean voided midstream urine sample is collected in a
sterile leak-proof container.
• Sample container is labeled, request form filled and send
along with the sample to the laboratory within an hour or
two.
• If delay of more than 1-2 hours is there, the sample
should be refrigerated.
• Boric acid can be used as a preservative to prevent the
change in count of bacteria in the sample.

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Dr Praveg Gupta MD 34
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 QUANTITATIVE AND SEMIQUANTITATIVE
METHODS
• Quantitative culture:
• Serial ten fold dilutions of urine are tested by the pour
plate or surface culture methods. Complicated method.

• Semiquantitative culture:
• Fixed volume of urine placed on a noninhibitory medium
like blood agar with a standard loop for quantitative
measurement.
• Another loopful of urine placed on a indicator medium like
MacConkey agar for presumptive diagnosis of causative
organism.

• Culture is followed by biochemical tests etc for

identification of the pathogen.
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DIARRHEA CAUSING E.COLI
• EPEC = ENTEROPATHOGENIC E.COLI

• ETEC = ENTEROTOXIGENIC E.COLI

• EIEC = ENTEROINVASIVE E.COLI

• EHEC = ENTEROHAEMORRHAGIC E.COLI

• EAEC = ENTEROAGGREGATIVE E.COLI

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 EPEC = ENTEROPATHOGENIC E.COLI

• Associated with diarrhea in infants and children.

• Institutional outbreaks, sporadic diarrhea.

• Do not produce enterotoxins.

• Not invasive.

• M/A: Attach to mucosa of upper small intestine, cause

disruption of brush border microvilli.

• Enter adherent E.coli is another name given to them

because they can adhere to HEp-2 cells.
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EPEC brush border disruption

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 ETEC = ENTEROTOXIGENIC E.COLI

• Endemic in developing countries in tropics, all age groups.

• Mild watery diarrhea to fatal disease indistinguishable
from cholera.

• Persons from developed countries visiting endemic areas

often suffer from ETEC diarrhea – a condition known as
TRAVELER’S DIARRHEA.

• Adhere to intestinal epithelium via fimbrial or colonization

factor antigens (CFA I,II,III,IV,etc.).
• Produce LT or ST or both.

• Diagnosis done by demonstration of the toxin.

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TRAVELER’S DIARRHOEA

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ETEC TRAVELER’S DIARRHOEA

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 EIEC = ENTEROINVASIVE E.COLI
• Resemble Shigella
• Many are nonmotile, do not ferment lactose or
ferment it late with only acid production, and do not
form lysine decarboxylase.

• Many of these show O antigen cross reactivity with

Shigella.
• Earlier names given – Shigella alkalescens, Shigella
dispar – and were grouped under Alkalescens-Dispar
group.

• Named EIEC because they have the capacity to invade

interstitial epithelial cells in vivo and penetrate HeLa cells
in tissue culture. Dr Praveg Gupta MD 43
• Clinically EIEC infection resembles shigellosis, ranging
from mild diarrhea to frank dysentery.

 Sereny test:
• Instillation of a suspension of freshly isolated EIEC or
Shigella into the eyes of guinea pigs leads to
mucopurulent conjunctivitis and severe keratitis.
• Mice can also be used.

 Cell Penetration in HeLa or HEP-2 cells.

 Plasmid detection:

 VMA ELISA: The plasmid codes for outer membrane

antigens called the virulence marker antigens (VMA)
which can be detected by the ELISA (VMA ELISA) test.
Dr Praveg Gupta MD 44
 EHEC = ENTEROHAEMORRHAGIC
E.COLI
• Produce VT
• Mild diarrhea to fatal hemorrhagic colitis and
hemorrhagic uremic syndrome (HUS) particularly in
young children and elderly.
• Primary target of VT = vascular endothelial cells.
• O157:H7, O26:H1 etc
• The disease may occur sporadically or as outbreaks
of food poisoning.
• Changing lifestyle and eating habits.
• Salad vegetables such as radish and alfalfa sprouts, in
which bacteria were found beneath the skin and in the
deeper tissues.
• Diagnosis: demonstration of VT.
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EHEC ATTACKS VASCULAR ENDOTHELIAL
CELLS, ALSO PRODUCES VT

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EAEC = ENTEROAGGREGATIVE E.COLI

• Appear aggregated in a stacked brick formation on

Hep-2 cells or glass.

• They have been associated with persistent diarrhea,

especially in developing countries.

• They form a LMW heat stable enterotoxin called EAST1

(enter aggregative heat stable enterotoxin-1).

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EAEC FORM STACKED BRICK LIKE
FORMATION

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• PYOGENIC INFECTIONS:
• E.coli form the most common cause of intra-abdominal
infections, such as peritonitis and abscess resulting from
spillage of bowel contents.
• Pyogenic infections in the perianal area.
• Neonatal meningitis

• SEPTICAEMIA:
• Blood stream invasion by E.coli may lead to fatal
conditions like septic shock and systemic
inflammatory response syndrome (SIDS).

• HeLa = Human carcinoma of cervix cell line (named after a lady

named Hela)
• HEP-2 = Human epithelioma of larynx cell line
• Vero = vervet monkey kidney cell line
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KLEBSIELLA

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 INTRODUCTION
• GENUS KLEBSIELLA:
• K.pneumoniae
• K.ozaenae
• K.rhinoscleromatis
• K.oxytoca etc.

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 GENERAL FEATURES
• Non-motile

• Capsulated

• Grow on OM forming large dome shaped mucoid

colonies.

• Short plump straight rods.

• Capsular halo seen prominently in gram stain.

• Commensals, saprophytes.
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 LARGE DOME SHAPED MUCOID
COLONIES AND CAPSULAR HALO

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 KLEBSIELLA PNEUMONIAE
(FRIEDLANDER’S BACILLUS, BACILLUS MUCOSUS
CAPSULATUS)
• Sugar fermentation – acid + gas
• IMViC --++
• Urease +

• Second most populous member of aerobic bacterial flora

of the intestine.

• Important cause of nosocomial infections.

• Pneumonia, UTI, pyogenic infections, septicemia, and

rarely diarrhea.

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 KLEBSIELLA PNEUMONIA
• Serious disease with high case fatality.

• Middle age or older persons.

• Alcoholism, chronic bronchopulmonary disease, diabetes.

• Massive mucoid inflammatory exudate of lobar or lobular

distribution, involving one or more lobes of the lung.

• Necrosis and abscess formation.

• Serotypes 1, 2, 3.
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KLEBSIELLA - MICROSCOPY

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KLEBSIELLA - on CULTURE

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Klebsiella on Nutrient agar and Blood agar

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 VIRULENCE FACTORS
• CAPSULE: Mucoid capsule is ant phagocytic and acts
as a major virulence factor.

• PLASMID EXCHANGE: Klebsiella participates in

exchange of plasmids with other Enterobacteriaceae.
The exchange of plasmid is presumed to be the basis
for two constant characteristics of Klebsiella species.
a. Antibiotic resistance
Many strains are highly resistant to most antibiotics.
b. Toxins
Some Klebsiella strains carry plasmids that code for
toxins similar to heat labile and heat stable exotoxins of
E.coli.
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 CLINICAL SYNDROME
1. PNEUMONIA

• K. pneumoniae is found in 10% normal individuals as

normal flora of respiratory tract.

• Pneumonia in diabetics, alcoholics and

immunocompromised patients.

• Lung abscess

• Lobar pneumonia – necrotic destruction of the alveolar

spaces, cavity formation and production of thick blood
tinged viscus sputum. Prognosis is grave with 50%
mortality.
Dr Praveg Gupta MD 60
2. URINARY TRACT INFECTIONS

3. SEPTICAEMIA

4. WOUND INFECTION

5. MENINGITIS

6. EPIDEMIC DIARRHOEA – in newborns.

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 LABORATORY DIAGNOSIS

• Specimen: urine, pus, blood etc. depending on the site of

infection.

• Culture: It grows on MacConkey and Blood agar media.

Colonies appear mucoid and pink in MacConkey agar
media.

• Biochemical: they ferment glucose and lactose producing

acid and gas.

• Typing: based on about 90 capsular (K) antigens there

are three types – K2, K3 and K21.
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 TRIBE
PROTEAE

Dr Praveg Gupta MD 63
 GENUS PROTEUS
• Lactose non fermenters.
• The name proteus refers to their pleomorphism, after
the greek god proteus who could assume any shape.

• Urease +
• PPA +
• Noncapsulated, pleomorphic, motile rods.

• Weil Felix observed that flagellated strains growing on

agar formed a thin surface film resembling the mist
produced by breathing on glass and named this variety
the Haunch form (haunch=film of breath).
• Nonflagellated strains did not form, so called Ohne
Haunch.
• Hence O = somatic antigen, H = flagellar antigen.
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Dr Praveg Gupta MD 65
 CULTURE CHARACTERISTICS

• Putrefactive (fishy or seminal) odor

• Swarming observed on NA, BA

• Pale colonies on Mac and DCA and no swarming

• Indole + (except pmi)

• H2S + (pmi and pvu)

• MR +, VP –
• Citrate + (except M morganii)

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• Habitat: widely prevalent in nature, commensals,
saprophytes, opportunistic pathogens.

 Swarming: wavy growth, thin filmy layer in concentric

circles shown by p.vulgaris and p.mirabilis in culture
plates.

 To inhibit swarming:
• 6% agar,
• sodium azide(1:500),
• alcohol(5-6%),
• sulphonamide,
• chloral hydrate(1:500),
• surface active agents or
• boric acid(1:1000).
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SWARMING OF PROTEUS ON NUTRIENT AGAR AND
BLOOD AGAR

Dr Praveg Gupta MD 68
Swarming a Distinguishing
Character in Proteus

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 CLINICAL SYNDROME
• URINARY TRACT INFECTIONS

• Urease producing organisms cause liberation of

ammonia which raises the pH of the urine. Alkalized
urine favors solubility of calcium that creates
environment favorable for deposition of calcium and
magnesium salts and the formation of kidney stones.

• Proteus infections tend to occur in patients in obstructive

lesions of urinary tract following diagnostic
instrumentation or during prolonged catheterization
(exogenous infection).

Dr Praveg Gupta MD 70
• WOUND INFECTIONS
• SEPTICAEMIA
• ACUTE OTITIS MEDIA

• LABORATORY DIAGNOSIS
• Choice of Specimen
• Collection and transport
• Processing

Dr Praveg Gupta MD 71
 WEIL FELIX REACTION
• They observed that certain nonmotile strains of proteus
were agglutinated with sera from typhus fever patients.

• This heterophile agglutination due to sharing of a

carbohydrate hapten by certain strains of proteus and
rickettsia forms basis of weil Felix reaction for Dx of
certain rickettsial infections.

• Nonmotile proteus strains OX2, OX19 (P.vulgaris) and

OXK (P.mirabilis) used in agglutination test.

Dr Praveg Gupta MD 72
 QUESTIONS
• Which E.coli causes institutional outbreaks of diarrhea in
small children?

• Which E.coli causes traveler’s diarrhea?

• Which sample should be collected for investigating a

case of Urinary tract infection?

• What is Kass’ concept of significant bacteriuria?

• Which E.coli is associated with hemolytic uremic

syndrome?

• Consumption of radish and alfalfa sprouts is associated

with which Diarrhoeagenic
Dr PravegE.coli?
Gupta MD 73
• Describe the colony of Klebsiella pneumoniae.

• Why is proteus named so?

• Describe key biochemical characteristics of proteus.

• What is swarming? How can it be inhibited?

• Which UTI organism can predispose to kidney stones?

• Which diarrhea causing E.coli is associated with

dysentery?

Dr Praveg Gupta MD 74
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