E.

Coli , Klebsiella

Dr. R.K.Kalyan
Professor
Microbiology
KGMU, Lko
 Learning objectives
The students will be able to answer the following questions:

 Describe morphology and antigens

 Describe Pathogenesis & Clinical features

 Choose appropriate lab diagnosis and interpret the results

 Describe prevention and treatment

 Enterobacteriaceae
 Family Characters (General Properties)
 Gram-negative bacilli or cocobacilli
 Non sporing, non acid fast
 ™
Aerobes and facultative anaerobes, N
™on fastidious
 Ferment glucose to produce acid with or without gas
 ™
Reduce nitrate to nitrite
 ™
Catalase positive, oxidase negative
 ™
Motile with peritrichous flagella, or non-motile
 Mostly commensals in human intestine - Coliform
bacilli
 Classification: Oldest method based on
their action on lactose
Groups Lactose Colonies on MacConkey Examples
fermentation agar
Lactose fermenters (LF)- Ferment lactose Produce pink colored Escherichia, Klebsiella
all are coliform bacilli producing acid colonies, Citrobacter
(acid changes the color of
neutral red indicator to pink)

Non lactose fermenters Do not ferment Produce pale or colorless Salmonella, Shigella
(NLF) lactose colonies Proteus, Morganella,
Providencia and Yersinia

Late lactose fermenters Ferment lactose after At 24 hrs incubation- Shigella sonnei
(LLF or previously called 2-8 days of produce pale or colorless
as paracolon bacilli incubation colonies,
After 2 days- produce pink
color colonies
Classification: Common morphogical, biochemical and similar
DNA base compositions. Bergey’s manual, Kauffmann, Edwards-
Ewing
Ewing’s Classification: Family is classified into its major
subdivisions, groups or tribe-genera-subgenera-species-types-
biotypes, serotypes, bacteriophage types, colicin types

Tribe Genus
Tribe I-Escherichieae Escherichia, Shigella
Tribe II-Edwardsielleae Edwardsiella
Tribe III-Salmonelleae Salmonella
Tribe IV-Citrobactereae Citrobacter
Tribe V- Klebsiellaae Klebsiella, Enterobacter,
Hafnia, Serratia, Pantoea
Tribe VI-Proteeae Proteus, Morgenella,
Providencia
Tribe VII- Yersinieae Yersinia
ESCHERICHIA COLI
 Genus named after Escherich who first isolated the
bacilli under the name Bacterium coli commune in
1885

 Most common aerobe harboured in gut of humans and

animals

 Detection of the thermotolerant E. coli (survives at

44°C) in drinking water → recent contamination with
human or animal feces

 ™
Other species are less important as human pathogens
- E. fergusonii, E. hermannii and E. vulneris
Morphology
 E.coli is a GNB, 1-3µm x 0.4-0.7µm

 Most strains are motile by peritrichate

flagellae

 Non- sporing and non-capsulated

 Culture
 Aerobic and facultative anaerobe and grows on
ordinary culture medium at 37ºC (10-40ºC) in
18-24 hrs

 MacConkey’s medium- pink, circular, moist,

smooth, with entire margin , non mucoid
colonies
 Some strains show β- haemolysis on BA media

 In liquid uniform turbidity

Biochemical Reaction
 They ferment most of the sugars ( glucose, lactose,
mannitol, maltose) with acid and gas

 Typical strains do not ferment sucrose

 Indole and methyl red (MR) reaction are positive but

Voges-Proskauer (VP) and citrate utilisation tests are
negative (IMVic++--)

 Urease –ve, Gelatin not liquified, H2S not formed

 No growth in KCN medium

 Virulence factors of E. coli
Two types of virulence factors of Esch.coli have identified
1.Surface antigens
i.Somatic Ag (O)
ii. Flagellar (H)
iii.Capsular antigens (K)
iv. Fimbrial antigen

2. Toxins: enterotoxin, haemolysin and verocytotoxin

 Antigenic structure: Serotyping of E.coli based on
presence of O, k, and H antigens detected by
agglutination reactions
Somatic or O antigen:
 Most important virulence factor → endotoxic activity, Protects from
phagocytosis and bactericidal effect of complement

- Lipopolysaccharide (LPS) antigen of cell wall, heat-stable

- Occasionally, it cross reacts with O antigens of other genera of

enterobacteriaceae( Citrobacter, Salmonella, Shigella, and Yersinia

- Early O serotypes - commensals of intestine- 1,2,3 etc

- Late O serotypes - diarrhea producing strains 26,55,111 etc

- More than 173 O seotypes

Virulence factors of E. coli
 Flagellar or H antigen (H from Hauch, meaning film of breath)
- These are thermolabile and 75 H antigens
- Heat labile, monophasic
- Motility contributing to virulence
 Capsular or K antigen
(K for Kapsel, German for capsule)-
- Polysaccharide capsular antigen present on the envelope or
microcapsule
- They cause ‘O’ inagglutinability by homologous antigen
- Expressed by some strains only - neonatal meningitis,
pyelonephritis and septicemia
- Most strains of intestinal E.coli do not possess K Ags.
- Encloses O antigen → inagglutinable by the O antiserum
- 103 K antigens are described
- Inhibits Phagocytosis
 Virulence factors of E. coli
 Fimbrial antigen (pilus) - organ of adhesion
 Thermolabile proteins and heating the organisms at 100ºC
leads to detachment of fimbriae
 Type I fimbriae mediate adhesion of bacterium to cells that
contains mannose residue
 Adhesions enhances bacterial pathogenicity -UTI
 CFA (colonization factor antigen): enterotoxigenic E. Coli
 Mannose resistant fimbriae (e.g. P, M, S, F1C and Dr
fimbriae):
- Hemagglutinate with RBCs that is not inhibited by mannose
- Expressed by uropathogenic E. coli and role in diarrhoeal ds.
 P fimbriae bind specifically to the P blood group antigens
present on human RBCs and uroepithelial cells
 Antigenic types
 On the basis of O antigen, E.coli has been divided into
a number of O groups

 Each O group divided into subgroups on the basis of K

antigens

 Each of these subgroups includes strains with

different H antigens

 Thus antigenic pattern of a strain is recorded as the

number of the particular antigen it carries

 E.g O111:K58:H12.
 Resistance
 E.coli is excreted in faeces of human and animals
and contaminate soil and water

 It is killed by moist heat at 60ºC usually within 30

minutes

 It can be killed by 0.5-1 part per million (ppm

chlorine in water

 It can survive for several days in soil, water, dust

and air
Toxins
1.Enterotoxins: produced by enterotoxigenic strains of E.coli
(ETEC).diarrheagenic strains of E. coli
- Heat labile toxin(LT) and heat stable toxin(ST) and verocytotoxin

2.‰
Hemolysins: virulent strains of E. coli (especially pyelonephritis
strains)
- Can lyse erythrocytes of some species
- A large proportion of E.coli recovered from extra-intestinal
lesion of man

3. Verocytotoxin( VT)

4.‰
Cytotoxic necrotizing factor 1 (CNF1) and secreted
autotransporter toxin (SAT): Cytotoxic to bladder and kidney cells

 Siderophores (i.e. aerobactin)—Helps in iron uptake

1. Heat labile enterotoxin
LT (heat-labile toxin)
 Produced by: Enterotoxigenic E. Coli
 Plasmid coded, Resembles cholera toxin but less potent
 LT is composed of one enzymatically active polypeptide A
( A for active) and 5 identical B (B for binding) subunits)
 Mechanism of action:
- Subunit B:Binds to GM1 ganglioside receptors on
intestinal epithelium →A fragment is internalized and
cleaved into A1 and A2 peptides
 LT (heat-labile toxin)
- Fragment A:
- Fragment A2 helps in tethering A and B subunits
together
- Fragment A1 - active fragment , causes ADP
ribosylation of G protein → upregulates activity of
adenylate cyclase → intracellular accumulation of
cAMP → increased outflow of water and electrolytes
into the gut lumen → diarrhea
Detection of LT:
 Toxin detection: latex agglutination, ELISA
 Molecular methods: PCR detecting gene coding for
LT
 ST (heat-stable toxin)

 Produced by: Enterotoxigenic E. Coli

 Plasmid-coded
 ST is of two types: ST-I and ST-II
 Mechanism of action:
- ST-I: Binds to the guanylate cyclase C → increased
production of cGMP → fluid accumulation in gut lumen
→ diarrhea
- ST-II: causes fluid accumulation by an unknown
mechanism
 Detection of ST: Same as for LT
 Verocytotoxin or Shiga-like toxin

 Produced by:
- Enterohemorrhagic E. Coli
- Bacteriophage-coded
 Cytotoxic to Vero cell lines,
 Also called Shiga-like toxin as it resembles
Shiga toxin in its structure and function
 Verocytotoxin or Shiga-like toxin
 Mechanism of action:
- Fragment B binds to a globotriosyl ceramide (Gb3)
receptor on intestinal epithelium
- Fragment A - Active fragment. Inhibits protein
synthesis
 Detection of VT:
 Serologically—Latex agglutination, ELISA
 Molecular methods—using specific DNA probe
 Cytotoxicity on Vero and HeLa cell lines
 Clinical Manifestations
 one of the most common pathogen encountered
clinically
 ™
Urinary tract infection (UTI): uropathogenic E. coli
(UPEC)
 Diarrhea: Six types of diarrheagenic E. Coli
1. Enteropathogenic E. coli (EPEC)
2. Enterotoxigenic E. coli (ETEC)
3. Enteroinvasive E. coli (EIEC)
4. Enterohemorrhagic E. coli (EHEC)
5. Enteroaggregative E. coli (EAEC)
6. Diffusely adherent E. coli (DAEC)
 Clinical Manifestations

 „
Abdominal infections: Commonest cause of
primary and secondary bacterial peritonitis
 Visceral abscesses - hepatic abscess
 Pneumonia in hospitalized patients— VAP
„
 Meningitis (especially neonatal)
„
 Wound and soft tissue infection - cellulitis and
„
infection of wounds
 Osteomyelitis, Endovascular infection and
bacteremia
 Laboratory Diagnosis – Specimen
collection
Specimens collected Disease
Pus, exudates and wound Cellulitis or pyogenic wound
swab infection
Urine UTI
Stool Diarrhea
CSF Meningitis
Peritoneal exudate Peritonitis
Sputum Pneumonia
Tracheal aspirate Ventilator associated
pneumonia
Blood Bacteremia
 Laboratory Diagnosis

 Direct Microscopy – Gram

negative Bacilli
 Culture: Aerobe and
facultative anaerobe,
nonfastidious
- Blood agar: Colonies are
big, circular, gray, moist and
occasionally β hemolytic
Disclaimer: This image for educational
- MacConkey agar: pink due purpose only not for commercial activity

to lactose fermentation
 Laboratory Diagnosis
 Liquid medium - uniform
turbidity
 Culture smear and motility
testing: Scattered gram-
negative bacilli
 Hanging drop – Motile
bacilli
Disclaimer: This image for educational
purpose only not for commercial activity
 Biochemical Tests
- Catalase positive and
oxidase negative  ‰
Sugar fermentation test:
- Nitrate is reduced to ferments most of the
nitrite sugars, such as glucose,
 ICUT tests:
‰ lactose, mannitol, maltose
(but not sucrose), with
- Indole test: Positive
production of acid and
- Citrate & Urease test: gas.
Negative
 ‰
MR (methyl red) test:
 Triple sugar iron agar) Positive
test: Shows acid/acid,
 ‰
VP (Voges-Proskauer)
gas present, H2S
test: Negative
absent
Laboratory Diagnosis of UPEC
 Specimen Collection
 ™
Clean voided midstream urine: Commonest
specimen - collected after properly cleaning
urethral meatus or glans
 Suprapubic aspiration of urine from the bladder:
™
most ideal specimen - for patients in coma or infants
 Catheterized patients - from the catheter tube
™
(after clamping and disinfecting); but not from the
uro bag
 Transport
 Processed immediately. Expected delay-
refrigerator or adding boric acid, glycerol or
formate
 Direct Examination & Screening Tests
 ™
Wet mount examination: Pyuria of more than 8 pus
cells/mm3 or 4 lakh pus cells excreted in urine/hour is
significant
 Leukocyte esterase test: Rapid and cheaper method
™
 Nitrate reduction test (Griess test)
™
 Gram staining of urine is not a reliable indicator
™
as—
- Bacterial count in urine is usually low
- Pus cells rapidly deteriorate in urine
- Limited to pyelonephritis and invasive UTI - count of ≥1
bacteria/oil immersion field is significant
 ™
Culture media: MacConkey agar and blood
agar or CLED agar
 ™
Kass concept of significant bacteriuria:
- A count of ≥105 colony forming units
(CFU)/mL of urine is considered as significant
→ Indicates infection „
- Count between 104 to 105 CFU/mL indicates
doubtful significance - clinically correlated
 Low count of ≤ 104 CFU/mL - Commensal bacteria due to
contamination during voiding. Low counts may be significant :
- Patient on antibiotic or on diuretic treatment
- Infection with some gram-positive organisms such as S. aureus and
Candida
- Pyelonephritis and acute urethral syndrome
- Sample taken by suprapubic aspiration
 Quantitative culture: Semi-quantitative method - standardized
™
loop technique
 Q
„ uantitative method such as pour plate method.
 Diarrhea (Diarrheagenic E. coli)

 Diarrheagenic E. coli are antigenically distinct

from the commensal E. coli which colonize the
intestine
 Only few serotypes of E. coli which express the
enterotoxin or other virulence mechanisms can
cause diarrhea
 Six types of diarrheagenic E. coli.
 Enteropathogenic E. coli (EPEC)
 Causes infantile diarrhea (outbreaks) and
occasionally sporadic diarrhea in adults
 ™
Nontoxigenic and noninvasive
 ™
Mechanism of diarrhea:
- Adhesion to intestinal mucosa mediated by plasmid
coded bundle-forming pili
 „
Attaching and effacing lesions: leads to disruption
of brush border epithelium causing increased
secretion and watery diarrhea
 Enterotoxigenic E. coli (ETEC)
 Most common cause of traveler’s diarrhea (25–75% )
 Acute watery diarrhea in infants and adults
 Common serotypes—O6, O8, O15, O25, O27, O153,
O159, etc.
 Toxigenic, but not invasive
 Pathogenesis of ETEC is by:
™
 Attachment to intestinal mucosa mediated by fimbrial
„
protein colonization factor antigen (CFA)
 Toxin production—(1) heat-labile toxin or LT (acts by
„
↑cAMP), (2) heat-stable toxin or ST (acts by ↑cGMP)
 Enteroinvasive E. coli (EIEC)
 Common serotypes - O28, O112, O114, O124, O136,
O143,O144,O152 ,O164
 Pathogenesis: Invasive
™
- Mediated by a plasmid-coded antigen called virulence
marker antigen (VMA)
- Biochemically, genetically & pathogenically related to
Shigella
 Manifestations: Ulceration of bowel, dysentery
™
 Diagnosis: Detection of VMA by ELISA
™
- HeLa cell invasion assay, „
„ DNA probes to screen faeces
- Sereny test; On instillation into the eyes of guinea pigs,
EIEC cause keratoconjunctivitis, no longer used.
 Enterohemorrhagic E. coli
(EHEC)

 ™
Serotypes associated with EHEC are: O157:H7 (most
common serotype)
 Other serotypes - O26:H11, O6, O55, O91, O103, O111 &
„
O113
 Transmitted by contaminated food, i.e. consumption of
™
lettuce, spinach, sprouts and undercooked ground beef
 Prevalent mainly in industrialized countries
™
 Low infective dose: Few organisms (<102 bacilli) are
™
required to initiate the infection
 Pathogenesis: secretes verocytotoxin or Shiga-like toxin
™
 Shiga-like Toxin
 Mechanism of action: inhibits protein synthesis by
inhibiting the 28S subunit of 60S ribosome.
 Stx2 is more commonly associated with HUS than Stx1
‰
 Manifestations: predilection for endothelium
™
→capillary microangiopathy
 H
„ emorrhagic colitis: gross bloody diarrhea,
abdominal pain and fecal leukocytosis but no fever
 Hemorrhagic uremic syndrome (HUS): injury to
„
small vessels of the kidney and brain→ bloody
diarrhea, thrombocytopenia, renal failure and
encephalopathy but without fever
 Diagnosis:
 „
Sorbitol MacConkey agar: Unlike other E. coli,
does not ferment sorbitol and produces pale
colonies

 „
Toxin detection:
 •
Demonstration of cytotoxicity in Vero cell lines
(gold standard method)
 •
Fecal toxin detection by ELISA or rapid tests
 „
PCR - to differentiate genes coding for Stx1 and
Stx2
Enteroaggregative E. coli (EAEC)
 Adheres to HEp-2 cells in a stacked-brick fashion
 Most strains are “O” untypeable but “H” typeable
 ™
Pathogenesis:
 „
Intestinal colonization mediated by aggregative
adhesion fimbriae I
 EAST 1 toxin
 ™
Manifestations: Persistent and acute diarrhea
 E. coli O104: H4 - enteroaggregative strain that has
caused major outbreaks in Germany in 2011. Also
produces Shiga-like toxin and can cause HUS
 Treatment E. coli
 Extra-intestinal E. coli
 ‰
Based upon antimicrobial susceptibility test report
 ‰
Hospital strains mostly MDR. Often produce ESBLs
or AmpC β-lactamases →resistant to most β-
lactams except carbapenems
 ‰
Carbapenems, amikacin or BL/BLIs - agents of
choice for hospital acquired MDR E. coli isolates
 Extra-intestinal E. Coli
 Carbapenem resistant isolates ‰
- Polymyxins,
fosfomycin or tigecycline

 Diarrheagenic E. Coli - fluid replacement

- Antimicrobials to be avoided
KLEBSIELLEAE
 Genera Klebsiella, Enterobacter, Hafnia and Serratia
differ from all other tribes being VP positive but MR
negative

 Klebsiella - found as commensals in human intestines

and as saprophytes in soil

 Genus Klebsiella has three species—K. Pneumoniae, K.

Oxytoca and K.granulomatis
 K.pneumoniae: 3 Subspp. Peumoniae, ozaenae and
rhinoscleromatis

 Lactose fermenters

 Non-motile and capsulated

 Morphology
 Short , coccobacilli, Gram negative, capsulated, non
motile bacilli
 Size 1-2 µm x 0.5-0.8 µm
 Culture: MA- Colonies are large, mucoid, LF,

Biochemicals;
Ferments sugar (G,L,S,M,) with production of acid and
gas
Urease positive, indole-ve, VP positive,, citrate utilizing(
IMViC --++)
 Antigenic structure
1. Capsular (K) antigen: on the basis of capsular
antigens, Klebsiella classified into 80 (1-80)
serotypes.
• Identification of capsular antigens usually done by
capsular swelling reaction with capsular
antiserum
2. Somatic (O) antigen: Klebsiella contains five (01-
05) different somatic or O antigens in various
combinations
 Methods of typing
 Phage typing,biotyping, bacteriocin( klebocin or
pneumocin) typing and resistotyping

 Many Klebsiella strains produce bacteriocins k/a

Klebocins or pneumocins which show a narrow range
of activity on other Klebsiella strains

 Klebocin typing and capsular serotyping together may

be very useful for epidemiological studies
 Pathogenesis
❖ Klebsiella pneumoniae subspecies pneumoniae:
- Most pathogenic
- Severe lobar pneumonia - destructive with production
of thick, mucoid, brick red sputum
- Urinary tract infections, meningitis (neonates),
septicemia and pyogenic infections such as abscesses
and wound infections
 Colonizes the oropharynx of hospitalized patients
„
 Common cause of nosocomial infections
 Most hospital strains - multidrug resistant
 Pathogenesis

❖ K. pneumoniae subspecies ozaenae

- Atrophic rhinitis (or ozena) -foul smelling nasal
discharge
- Biochemically inactive
❖ K. pneumoniae subspecies rhinoscleromatis
- Rhinoscleroma - chronic granulomatous hypertrophy
of the nose
- South eastern Europe, India and in Central America
- Biochemically inactive
 Laboratory Diagnosis

 ™
Gram staining: short,
plump, straight
capsulated gram-
negative rods

Disclaimer: This image for educational purpose only not

for commercial activity
 Klebsiella
 ™
Culture:
 MacConkey agar - large
dome shaped mucoid (due
to capsule) sticky, pink
colour, lactose fermenting
colonies

Disclaimer: This image for educational

purpose only not for commercial activity
 Biochemical identification:
 ICUT test:
-Indole test: Negative
- Citrate test & Urease test: Positive
- Triple sugar iron agar test: Acid/acid, gas present,
H2S absent

 ‰
Sugar fermentation test: Ferments most of the sugars
glucose, lactose, mannitol, maltose (but not sucrose),
with production of acid and gas
 ‰
VP (Voges-Proskauer) test: Positive

 ‰
MR (methyl red) test: Negative

 K. oxytoca is biochemically similar to K. pneumoniae,

but differs from the latter by being indole positive

 Treatment: Most clinical isolates are MDR

- Guideline for treatment is same as that for E. coli

HAVE A NICE DAY

Disclaimer: This image for educational purpose

only not for commercial activity