This is the basic approach to cover the multi facets of E. coli with description of different pathotypes in detail

1. Understanding Colloquial
laboratory workhorse:
Escherichia coli
PANKAJ DHAKA,
Ph.D Scholar, P-1741
VPH, I.V.R.I
pankaj.dhaka2@gmail.com

2. HISTORY: Escherich’s great legacy to science
Escherich 1st described Bacterium coli commune in 1885
 Theodore Escherich (1857– 1911)
 Isolated from infant’s stool and
chose the designation Bacterium coli
commune (the common colon
bacterium)
 Renamed in his honour as
Escherichia coli in 1958 Judicial
Commission: “Conservation of the
Enterobacteriacea, ...”

3. Classification:
Genus : Escherichia
Family: Enterobacteriaceae
Order: Enterobacteriales
Class: Gammaproteobacteria
Phylum: Proteobacteria
Kingdom: Eubacteria
(Bergey’s Manual of Determinative Bacteriology, 1993)
 Gram-negative, rod-shaped, non-sporulating
 Facultative anaerobes of human & animal
intestine
 2.0-6.0 μm long & 1.1μm in diameter
 Motile with peritrichous flagella
(Bergey’s Manual of Determinative Bacteriology, 1993)

4. Colloquial laboratory workhorse
Biology’s premier model organism
•More known about this bacterium,
esp. K-12, than any other organism
• Escherichia coli- Most thoroughly understood
organism on earth
 Much of our knowledge of intermediary
metabolism, genetic recombination, DNA
replication, RNA transcription, and protein
synthesis, sorting…………… carried out in
E. coli and still carrying on………

5. The Good…..+
• Colonizes neonates shortly
after birth and remain with
us throughout life….
– Mostly nonpathogenic
– Coexisting in harmony
– Symbiotic relationship
– Synthesize cofactors
– Contribute to colonization
resistance against pathogenic
organisms
– Produces Vit. B12 & K
5
Most abundant facultative anaerobe present in the
intestine of humans and many other endothermic species

6. Escherichia coli : The enemy within
 Diarrheal illness- 2nd leading cause of U5MR, much
high in Africa and S. Asia (WHO, 2012); (Liu et al., 2012)
Rotavirus & E. coli- two most common etiological
agents of diarrhoea in developing countries (WHO, 2009)
 Globally, DEC are responsible for ≈ 300,000-500,000
U5MR; accounting around 33% of total infant’s death
(Fleckenstein et al., 2010)
Probiotic and pathogen!

7. More than humble resident of the gut ……….
The quintessential pleuripotential pathogen...
• Wide variety of illnesses in array
of species
• Target organs: GIT, meninges,
kidneys ……
• Outcomes:
– Diarrhea
– Dysentery
– Cholecystitis, UTI
– Peritonitis
– Pyelonephritis
– meningitis, HUS….

8. Breaking the Barrier Between
Commensalism and Pathogenicity
I am not a
cucumber
« commensal » E. coli can be also very bad guy… pathogenic to MDR

9. High number of different pathotypes
of E. coli due to genome plasticity
PATHOTYPES: A group of strains of a single species that cause a
common disease using a common set of virulence factors (Kaper et. al., 2004)
• GIT pathogens: Six categories
– enteropathogenic E. coli (EPEC)
– enterohaemorrhagic E. coli (EHEC)
– enterotoxigenic E. coli (ETEC)
– enteroaggregative E. coli (EAEC)
– enteroinvasive E. coli (EIEC)
– diffusely adherent E. coli (DAEC)
• Extraintestinal infections - ExPEC
• UTIs: Uropathogenic E. coli (UPEC).
• Meningitis & sepsis: meningitis-associated
E. coli (MNEC).
(Croxen & Finlay, 2010, Nat Rev Microbiol)

10. Patho-adaptive mutations
……More outbreaks….
• The core E. coli genome – only 20% of its avg. genome size (Rasko et al., 2008)
• More subtle patho-adaptive mutations-disease/Outbreak
• 2011: E. coli outbreak in Germany- >4,000 cases, 908 cases of HUS & >50
deaths, due to fenugreek seeds contaminated with the enteroaggregative
EHEC strain O104:H4 that produced Shiga toxins, imported from Egypt
(BFR, 2012)
Only 20% of the genome of E. coli (the 'core' genome) is highly conserved between strains

11. Jeckel and Hyde species…… can both coexist
peacefully with its host & cause devastating illness
HGT
What is responsible for this paradox?
-Conjugation
-Transformation
-Transduction
Mobile genetic elements
-Transposons
-Insertion sequences
-Bacteriophages & plasmids
New traits
Adaptation
 Fitness to bacteria
Virulence factors in humans
• Particular virulence gene combinations specific pathotypes of E. coli
• Each pathotype has a propensity to cause a limited variety of clinical syndromes

12. Complex relationships among pathotypes
 Share many virulence factors, single
clones can cause both meningitis and
urinary tract infections (Russo and Johnson,
2000)
 Among the UPEC, some strains share
with diffuse adhering E. coli (DAEC)
 DAEC strains recovered from individuals
with both urinary tract infections (UTIs) and
diarrhea (Germani et al., 1997)
 There are also reports of Stx producing
E. coli (STEC) strains causing UTI (Tarr et
al., 1996) and other extraintestinal
infections.

13. An emerging hybrid E. coli pathotype
+
EAEC
= EHEC O104:H4
Stx2 EaggEC Virulence plasmid
Colonization of the gut
Toxin production
Hemolytic-uremic
syndrome

14. Addition/Deletion…..New pathotypes
(Croxen et. al., 2013)

15. Pathogeneses of GIT pathotypes : An overview
(Croxen et. al., 2013)

16. Glimpse of pathogenesis
Pathotype Host(s) Site Disease(S) Resevoir(S)/sources Rx Adhesion Genetic Identifiers
(Croxen et. al., 2013)

17. EPEC: An introduction
• 1st pathotype of E. coli proved to be
associated with diarrhea- outbreak in a
pediatric nursery in London (UK)
by John Bray & colleagues, 1945
• Term ‘EPEC’: Neter et. al.,1955- linked
to childhood diarrhoea
• Serious threat to children < 2 years
• LEE +ve
– “typical” EPEC= +ve for E. coli
adherence factor plasmid (pEAF) and bfp
gene.
• Reservoir : Human only
– “atypical” EPEC: Highly heterogeneous
group, -ve for pEAF and bfp gene. Both
in human and animals
(Nataro and Kaper, 1998) Attaching and effacing (A/E) pathogens

18. EPEC: Pathogenesis
Adherence: Bundle forming pili (bfp);
tether individual bacteria  localized
adherence (LA)
Signal transduction and intimate
attachment- Bacterial signal proteins
injected into the epi. cell by means of a
Type III secretion system  changes to
the actin scaffold  destruction of the
microvilli
Pedestal-like structures are produced
through secretion of a conserved bacterial
receptor protein, Tir, via a type III
secretion system (T3SS)

19. Scanning electron micrographic (SEM) image of EPEC forming pedestals on
infected mammalian cells. Courtesy: Jorge Giron (University of Florida).

20. Normal microvilli Loss of microvilli
Uninfected Intestinal Cell Surface EPEC infected intestinal cells

21. Enterohaemorrhagic E. coli
• 1st recognised in 1982
• Common in developed countries
• “Sakai” strain caused Sakai/Osaka
outbreak in 1996
– >9000 cases, 12 deaths.
• Several other outbreaks of EHEC
(e.g. in California spinach, Sept 2006)
• Outbreaks usually linked to manure/
fecal contamination of food
• Commensal in cattle

22. EHEC: An introduction
 stx1 or stx2 gene typically acquired by a lambdoid bacteriophage qualifies it as
Shiga toxin-producing E. coli (STEC) or verocytotoxin producing E. coli (VTEC)
 EHEC is a subset of STEC: Mainly ssociation with hemorrhagic colitis (HC)
A/E lesion formation is caused by the
T3SS encoded by the LEE, which injects
small effector proteins into the host cell
STEC O157:H7 strains carry a plasmid
called pO157 that contains a catalase-peroxidase
gene (katP) and genes for
other virulence factors such as an
enterohemolysin (ehx) and toxB and espP
 stx1 HUS
 stx2  more severe sequele

23. • Stx binds to globotriaosylceramide receptor (Gb3) on the surface of endothelial
cells & is internalized through the retrograde pathway from Golgi apparatus & ER
and eventually to the cytoplasm
• STEC is able to cross the intestinal epithelium through microfold cells (Mcells)
and survive in macrophages  released into the bloodstream, where it can target
other organs (Etienne-Mesmin et. al., 2011)
• Cytolethal distending toxin (cdtABC) (Janka et al., 2003)
• The EHEC hemolysin (EHEC-hlyA or ehx) - a pore-forming toxin. Ehx was found
to be cytotoxic to endothelial cells and may contribute to the development of HUS
(Aldick et. al., 2007)
• Autotransporters in STEC is serine protease EspP. EspP is a multifunctional
protease that cleaves human coagulation factor V, pepsin A ( Brunder et. al., 1997)

24. EHEC virulence factors
Shiga-like Toxin
(aka SLT; Vero-Toxin; VT; Stx)
A/E Lesion
Type III secretion
plus pO157-encoded
ToxB

25. http://www.rpc.msoe.edu/cbm/smartteams/remote/
A subunit: cleaves rRNA
inhibits protein synthesis
B subunits: bind Gb3
Holotoxin

26. EHEC genetics of virulence
Shiga Toxin and T3SS effectors
encoded by bacteriophages

27. • Common serogroup O157:H7: Adulterant
in beef since 1994 in U.S
• USDA: 6 more EHEC serogroups
adulterants, i.e., O26, O45, O103, O111,
O121, and O145 (also known as “Big 6”)
• Transmission: Ruminants reservoir
• Faeco-oral route; low I.D ≈ 10-100 org.
• Form viable-but-nonculturable (VBNC) on
food and are still able to produce the Shiga
toxin  replicate & survive for long
periods of time on various food sources
(Dinu and Bach, 2011)
• Internalization inside spinach leaf tissue
(Saldaña et al., 2011)
• Asymptomatic shedders: person-to-person
transmission
TRANSMISSION

28. EHEC regulates both acid
resistance and the LEE genes
• Transcriptional regulator SdiA regulates both transcription of the LEE genes for A/E
lesion formation and the gad genes for acid resistance in cattle
• SdiA senses acyl-homoserine lactones (AHLs) produced by other bacteria in rumen
activate the gad genes - vital to acid resistance for the passage through the acidic
stomachs, and repress the LEE genes- prevent colonization within the rumen
• EHEC does not encounter AHLs beyond the rumen, alleviating the SdiA-mediated
repression of the LEE and allowing EHEC to colonize the RAJ (Hughes et al., 2010)

29. STEC beyond EHEC…
the German E. coli O104:H4 outbreak
• May-July 2011
• >4000 cases
• >40 deaths
• Link to sprouting seeds
• High risk of HUS
• Females particularly at risk

30. Take-away messages
• Infection still presents threat even in the most advanced societies
• Pathogens don’t bother with passports!
– Not a new strain: something similar seen in Germany ten years ago and in
Korea
– closest genome-sequenced strain was isolated from Central African
Republic in late 1990s, belongs to an enteroaggregative lineage
• German STEC probably comes from a lineage circulating in human
populations rather than from an animal source (unlike E. coli O157)
• Bacteria evolve quickly
– Virulence factors in E. coli can jump from one lineage to another on
mobile genetic elements
– Pathotypes can overlap and evolve
– Antibiotic resistance seen where no obvious prior use of antibiotics

31. Clinical considerations:
• Mild watery diarrhea  bloody diarrhea
(HC)  HUS
• I.P- for STEC O157:H7 is about 3 days
Symptoms
– Diarrhea
– Fever
– Abdominal cramping
– Aomiting
– Hemorrhagic colitis
– Longterm sequelae (in 20 to 40% of
patients) include cardiac complications,
neurological disorders, hypertension,
chronic renal disease, cognition and
behavior changes.
– It has been recommended that follow-up
assessments for late-onset sequelae be
done for at least 5 years (Rosales et. al., 2012)

32. Enterotoxigenic E. coli (ETEC)
• Major cause of traveler’s diarrhea &
endemic in underdeveloped countries
• Ability to produce either a heat-labile (LT)
or a heat-stable (ST) entero- toxin, & it
carries a diverse set of colonization factors
(CFs) for adherence to the intestinal epi.
• Swine industry is also adversely affected-induced
diarrhea in neonatal &
postweaning piglets
• Nearly 840 mn annual cases of ETEC in
developing countries (Wennerås and Erling, 2004)
• Transmission: fecal-oral route- from
contaminated food and drinking water
• I.D: relatively high i.e., between 10⁶-10⁸
org. (Levine et. al., 1979)
Countries at high risk for traveler’s diarrhea (CDC, 2007)

33. Heat-labile toxin (LT)
• Plasmid-encoded
heterohexamericholotoxin,
closely related to cholera toxin
• single A subunit, two domains
linked by disulfide bridge
– A1: active toxin molecule
– A2: helical anchor to B pentamer
• intact A not enzymatically
active until nicked to A1, A2
– A1 subunit released by reduction
of disulfide bond
• pentameric B subunit
– binds to GM1 gangliosides
centered in caveolae on host cell
surface
– triggers endocytosis of holotoxin

34. Heat-labile toxin (LT)
• Assembled as an AB5 toxin, LT is a large toxin that is about 80% identical
to the cholera toxin
• Briefly, the pentameric B subunits bind to GM1 gangliosides at lipid rafts
to deliver the catalytic A subunit inside the cell
• Once inside the cell, the A subunit is trafficked through ER (retrograde) and
delivered to the host cytoplasm, where it ADP-ribosylates Gs, inhibiting its
GTPase activity and increasing cyclic AMP levels
• cAMP opens the cystic fibrosis transmembrane receptor (CFTR), resulting
in electrolytes and fluid loss into the intestinal lumen
(Jobling and Holmes, 2012)

35. Heat-stable toxin (ST)
• Small cysteine-rich peptide secreted by ETEC
– can be boiled!
• binds to extracellular domain of guanylylcyclase C
– molecular mimicry: resembles endogenous ligand
guanylin
• activates intracellular catalytic domain of
guanylylcyclase
– intracellular accumulation of cGMP
– activates cGMP-dependent protein kinase II
– leads to phosphorylation of CFTR
• Cl- secretion and inhibition of NaCl absorption
leads to osmotic diarrhea

36. ETEC adhesins
CS3 fibrils
CFA 1 and III

37. Enteroaggregative E. coli
• Linked to persistent diarrhoea in
children
• Like ETEC strains bind to
enterocytes: do not invade
• Differ from ETEC strains
– do not adhere uniformly to mucosal
surface; form biofilms
– auto-aggregative: clump in small
aggregates (stacked-brick
appearance)
– relies on aggregative adherence
fimbriae (AAFs, related to Dr
family), dispersin
• Produce
– ST-like toxin EAST, but also
found in many commensals
– Autotransporters Pet and Pic

38. Pathogenesis:
Adherence to
the intestinal
mucosa
Biofilm formation
over enterocyte
surface
Release of toxins
Induction of proinflammatory
response
Mucosal toxicity & enhanced
intestinal secretion
(Cennimo et al., 2007)

39. Enteroinvasive E. coli (EIEC)
• Facultative i/c  bacillary dysentery
• Non-motile, lysine decarboxylase
negative & unable to ferment lactose
• Both the acquisition and loss of genes
 commensal E. coli with an
extracellular lifestyle  i/c pathogen
that is fully adapted to the diverse env.
challenges within its host
• Acquisition of the invasion plasmid
pINV and other virulence-associated,
mobile genetic element
• EIEC and Shigella are highly invasive
pathogens that use the intracellular
milieu of intestinal epithelial cells
(IECs) in the large intestine as their
replicative niche

40. • Penetration of epithelial barrier- through
M cells by transcytosis and macrophage
cell death by effector dependent
phagosomal escape and induction of
caspase I-dependent pyroptosis
• Invasion- by the ability of its effector
repertoire to subvert host cell signaling
pathways cell-to-cell spread
• Suppression of host immune response.
Host inflammatory responses, such as the
mitogen-activated protein kinase (MAPK)
and NF-B pathways, as well as cytokine
production, are targeted and dampened by
multiple effectors, including OspG, OspF,
OspB, OspZ, OspI, and IpaH
• Intra- and intercellular movement- T3S
effectors are responsible

41. Diffusely Adherent E. coli (DAEC)
• DAEC pathotype describes
diarrheagenic E. coli strains
that attach to cells but do not
fall into classical patterns of
adherence, such as localized
or A/E. They have now
emerged as a unique group
and are considered distinct
from other pathotypes
Distinctive adhesion patterns displayed by E. coli strains
on HeLa cells in the presence of D-mannose (A)
Localized adherence (LA)- EPEC strain (B) Aggregative
adherence (AA)- EAEC (C) Diffusely adherent E. coli-bacterial
cells scattered on the surface of host cell as well
as on the abiotic surface (D) Non-adherent E. coli

42. E. Coli leading cause of UTI
– Clinical significance
– Is the leading cause of urinary tract infections
which can lead to acute cystitis (bladder
infection) and pyelonephritis (kidney infection)
pks : a pathogenicity island present in the genome of E. coli responsable of
sepsis and urinary tract infections
This cluster of genes encodes the production of a
putative hybrid peptide-polyketide genotoxin : Colibactin
R1 N
H
O
OH
H
N
O
O
OH
N
H
O
H
N
O
O
SH
S H
N
N
O
O
COOR2
CONH2
pks cluster: 52 kb
Colibactin in silico predicted struture
Nougayrede et al Science 2006
The most important secreted virulence factor of UPEC  lipoprotein called α-haemolysin (HlyA) 
pore-forming toxin, which belongs to the family of RTX (repeats in toxin) toxins

43. Facts on UTI
UPEC is the cause of community-acquired UTIs and a large portion of nosocomial UTIs,
accounting for substantial medical costs and morbidity and mortality worldwide
Among Gram -ve bacteria, E. coli is most frequent pathogen inducing acute renal failure
Urological complications- E. coli is the most common clinical isolate
• Women suffer more than males Short urethra
• Other factors
Urethral obstruction,
Urinary stones
Congenital malformation's
Neurological disorders,
Catheterization , Cystoscopy
Usually cystitis is produced from fecal strains
entering urethra
(Bein et. al., 2012)

44. Diseases in animals

45. Diagnosis: Cultural techniques
 MacConkey agar: pH change- lactose fermentation
– E. coli colonies will appear red or pink on media
– Not all E. coli strains, particularly most EIEC and Shigella strains,
ferment lactose, so caution must be used when using this diagnostic
• EMB agar: a differential microbiological medium, which slightly inhibits the
growth of Gram-positive bacteria
– Rapid lactose fermentation produces acids, which lower the pH. This
encourages dye absorption by the colonies, which are now colored purple-black.
– Lactose non-fermenters may increase the pH by deamination of proteins.
This ensures that the dye is not absorbed. The colonies will be colorless.
– E. coli will give a distinctive metallic green sheen (due to the metachromatic
properties of the dyes, E. coli movement using flagella, and strong acid end-products
of fermentation)
– Sorbitol- MacConkey (SMAC) agar:
– EHEC O157:H7 isolates are unable to ferment sorbitol within a 24-h
period, they are easily distinguishable as clear colonies

46. Biochemical tests:
- Indole test- Positive (99% of E. coli strains)
 Single best test for differentiation from other
members of Enterobacteriaceae (Nataro and Kaper, 1998)
- IMViC reaction : + + - -
Methyl-Red Test Voges-Proskauer Test
Indole test (+)
Methyl-Red test (+)
Voges-Proskauer test (-)
Citrate Utilisation test (-)
Catalase test (+)
Carbohydrate fermentation test (+)
Indole Test
Citrate Utilisation Test
Catalase Test
Glucose & Lactose
fermentation Test

47. Serotyping
• Kauffman classification scheme
– O (somatic) polysaccharides and H (flagellar)
surface Ag.
– Molecular methods such as PCR of genes
involved in O-antigen biogenesis (e.g., wzx and
wzy genes) and of fliC for the H antigen, can
also be used to identify the serotype
– Currently, there are 174 E. coli O and 53 E. coli
H antigens ( DebRoy et. al., 2011)
Informative for certain pathotypes (e.g.,
O157:H7)
• Some isolates: untypeable or bear cross-reactivity
between antigens.
H antigen depends on variation in flagellin
O antigen depends on variation in LPS

48. Typing methods:
• For phylogenetic analysis outbreaks, and
surveillance investigations.
• Pulsed-field gel electrophoresis (PFGE) 
gold standard for typing  epidemiological
investigations to discriminate between
outbreak isolates
• Multilocus variable-number tandem repeat
analysis (MVLA)
• Multilocus sequence typing (MLST) - 
clonal complexes
• Molecular methods
• Higher discriminating ability
• Better characterization of the DEC
isolates (Dhanashree et al., 2012)
• Polymerase chain reaction (PCR)
• Used to detect virulent genes
(Toma et al., 2003)

49. Diagnosis: A glance

50. Treatment
– Antimicrobial therapy- E. coli was usually susceptible to a variety
of chemotherapeutic agents
– Scenario has changed rapidly: Drug resistant strains are
increasingly prevalent.
– It is essential to do susceptibility testing
– Treatment of patients with EHEC infections is not recommended
because it can increase the release of stx and actually trigger HUS
Drug resistance:

51. “Adopt the pace of nature: her secret is patience” - R. Emerson