STEC Flashcards
List some of the different variants of E. Coli
Enteropathogenic E. Coli (EPEC)
Enterotoxigenic E. Coli (ETEC)
Diffusely adherent E. Coli (DAEC)
Enterohaemorrhagic E. Coli (EHEC)
Enteroinvasive E. Coli (EIEC)
Enteroaggregrative E. Coli (EAEC)
Uropathogenic E. Coli (UPEC)
Neontal meningitis E. Coli (NMEC)
EPEC, ETEC and DAEC cause infections where in the body?
Colonise the small bowel and cause diarrhoea
EHEC and EIEC cause infections where?
They cause disease in the large bowel
EAEC infects what part of the body?
Can colonise both the small and large bowels
Where does UPEC cause infection
Uropathogenic E. Coli enters the urinary tract and travels to the bladder to cause cystitis and if left untreated can ascend durther into the kidneys to cause pyelonephritis
It can also spread to blood and cause urosepsis
Where does NMEC cause infection
It can cause septicaemia
Neonatal meningitis E. Coli can cross the blood brain barrier into the central nervous system and cause meningitis
What E. Coli strains colonise the small bowel and cause diarrhoea
EPEC
ETEC
DAEC
What E. Coli strains cause disease in the large bowel
EHEC
EIEC
What E. Coli strains colonoise both the small and large bowel
EAEC
What E. Coli strains cause septicaemia
UPEC
NMEC
Who are most at risk of urosepsis
Elderly women -> UTIs progress into septicaemia
How do the E.coli strains differ from one another
They differ in terms of pathogenicity
some inherit toxins such as shiga toxin in EAHEC
What is VTEC
Verotoxigenic E. Coli
What are the genes for VTEC, what do they encode
Stx1 and Stx2
Verotoxins encoded on bacteriophages
What are the genes for EHEC and what is their function
vtx1, vtx2, eae genes
Haemorrhagic colitis hence enterohaemorrhagic E. Coli
How was E. Coli traditionally serotyped?
Traditionally classified on basis of the reaction of antibodies with three types of antigens: O, K and H antigens
According to this method there is over 170 O, 103 K and 56 H antigens -> this is always increasin
A combination of O and H antigens have been identified to type strains
NB: these arent useful predictors of virulence
Give some examples of E. Coli strains named according to O, H and K antigens
O157:H7
O104:H4
O26
O103
O111
Where did verotoxigenic E. Coli get its name
VTEC comes from the old assay that was used to identify verotoxin producing e. coli
Vero monkey cells
What two organisms produce shiga toxin
Shigella dynsentriae type 1
Shiga toxin-producing E. Coli
Traditionally how was functionally active shiga toxins detected?
Using vero cell toxicity test
What strains will be vero cell toxicity test positive?
Verotoxin or verocytotoxin-producing E. Coli (VTEC)
What does shiga toxin cause?
Diarrhoea
Haemorrhagic colitis
Haemolytic uremic syndrome (HUS)
Enterohaemorrhagic E. Coli (EHES)
Technically speaking Shiga toxin could be produced by any E. Coli why is this?
Since the gene for it is transferred via bacteriophage so technically any strain can take it up
Talk about STEC/Shiga toxin E. Colli, what is it, how many different types are there
These produce 1 or more types of shiga toxin (stx):
- stx 1 or stx2
There are over 400 E. Coli serotypes which harbour stx genes
270 serotypes have been associated with clinical infection
- virulence may differ between strains
Genes are located on distinct phage elements - mobile genetic elements
Is stx1 or stx2 more toxigenic
Stx2 is 1000 times more toxic
What is the best studied strain of STEC
E. Coli O157:H7
When was O157:H7 first discovered
First recognised as a pathogen in 1982
It was identified as the cause of two outbreaks of bloody diarrhoea:
- fast food chains across several states on the west coast - undercooked burgers - caused 700 cases and 4 deaths
How did O157:H7 develop
There is lots of plasticity in the E.Coli genome -> theres a great ability to lose and acquire genetic material
Done through genomic islands and integrated prophages from bacteriophages
What did O157:H7 develop from, how did it evolve?
Prior to the initial outbreak it had never been isolated
It started of as an EPEC gene -> H7 was already present in enteropathogenic E. Coli first
EPEC then acquired a prophage which encoded the STX2 gene
PO157 plasmid was then collected
Plasmid encodes a haemolysin but lost the ability to fermen sorbitol and Bgluc
O polysaccharide was then changes
NB: we dont know what was the reservoir for all of these genetic changes
Give a quick breakdown on the development of O157:H7
Initially was an EPEC O55:H7
Stx phage was inherited -> EPEC O55:H7
Plasmid O157 inherited -> conversion to an EHEC O157:H7
Loss of abiliity to ferment sorbitol and Bglu to form the EHEC O157:H7 we know today
How is O157:H7 transmitted
There are multiple routes of transmission
Really low infectious dose needed
Direct contact and person to person spread
Cows are the reservoir -> spread to meat, unpasteurised foods or ready to eat foods, bodies of water etc -> ingestion by person - person to person spread
Outbreak associated with unpasteurised apple juice
What were the three main O157:H7 outbreaks studied in America
Spinach (SP)
Taco Bell (TB)
Taco John (TJ)
How do we detect polymorphisms in E. Coli
Lineage-specific polymorphism assay-6
Puts polymorphisms into lineages e.g. LI, LII etc
- there are three different lineages
Can be further broken down into Clades
- there are 8 different clades
How do lineages of O157:H7 vary from country to country?
Different lineages predominate in different countries e.g. L1 predominates in Canadda and US while LI/II predominates in australia and Argentina etc
How do Clades of O157 differ from contry to country
There are 8 different clades, 8 is hypervirulent
Clade 7 predominantes in Australia (92%) while clade 8 predominates in Argentina (81-91.4%)
In the netherlands the only clade identified is 8 (38.8%)
How many known differnet SNP genotypes are there for O157:H7?
39 SNP Genotypes
20% SNP difference at 96 loci
9 distinct clades
What is clade 8 of O157:H7 associated with?
Hypervirulence
but also clinical illness and more frequently reported blood diarrhoea
What are clades 2, 7, and 8 of O157:H7 associated with?
These are the clades associated with clinical illness
What are clades 2 and 8 of O157 associated with?
Clades 2 and 8 are associated with increased reports of bloody diarrhoea
What are the reservoirs for O157?
Generally considered to be cattle
Studies have recovered both O157 and non O157 from ruminants of both food producing and wild animals such as birds
Some animals can be super shedders
What are super shedders in terms of O157 reservoirs
Animals that shed greater than 10^4 CFU/g faeces
These animals are colonised at the recto-anal junction
These animals are more likely to spread the disease
Talk about O157 colonisation in animals
Some animals will be transiently culture positive
- short durations - few days
- not colonised at the RAJ mucosa
- passive shedders
- seen in cattle
- shed bacteria for an average of 1 month but less than 2 months
In rare cases animals can be colonised for a long time and can shed bacteria for 3-12 months or longer
Talk about non-O157 EHEC, what are they and how common are they?
Non-O157 EHEC infections are linked to the ‘Big 6’
- 70 to 75% are 1 of 6 specific strains
- 20 to 30% are other non-O157 STECs
These are becomig a lot more common
Rates ever increasing since bringing in molecular methods of typing EHECs
The disease association with these is unknown as research has focused on O157 but theyre all considered moderate risk compared to the high risk O157
What are the Big 6 Non-O157 EHECs
O26 - this is the most common in Ireland -> actually more common than O157
O45
O103
O111
O121
O145
Which of the non-O157 strains are most common
O26
O111
O103
O145
In this order -> O26 and O111 similar numbers
What factors contribute to the virulence of O157:H7
adhesion
LEE
Haemolysin (encoded by plasmid)
ToxB -> prophage
Mobile genetic elements:
- phages
- genomic islands
- plasmids
What is LEE in O157
Locus of enterocyte effacement
Its a pathogenicity island that inserts itself into the E. Coli genome
It allows for really unusual binding
It already existed in EPEC the E. Coli strain that O157 evolved from
What is special abobut the O157 adhesion to host epithelial cells?
Formation of attaching and effacing (A/E) lesions
Histopathological alteration of the intestine
Locus of enterocyte effacement (LEE) pathogenicity island -> unusual binding of EPEC
Unusual tight binding to epithelium cells
Alteration of the binding site
Need detailed descripion of tight binding of O157:H7 to epithelium
Talk about EHEC adherance to intestinal mucosa
EHEC can adhere to intestinal mucosa
EHEC has the eae gene which encodes intimin
eae = Attaching and effacing lesion
Intimin is used for the effacement of microvilli -> inimin binds closely and causes colonisation of the intestine
EHE colonises the intestinal mucosa and induces a characteristic histopathological lesion referred to as attaching and effacing A/E lesions
The geness responsible for A/E lesions map to 13 regions which have been designated the locus of enterocyte effacement (LEE)
What is eae
attaching and effacing lesion
Its a gene that encodes intimin
Its responsible for the effacement of microvilli in EHEC
What is an A/E lesion
A histopathological lesion characteristic to EHEC
Known as attaching and effacing lesion
Theyre encoded by genes found in LEE
Presence of AELs are associated with infection but bacteria cn still cause major disease without them
Talk about LEE expression
The presence of the LEE is stongly associate with disease
The LEE of E. Coli O157:H7 is conserved in EPEC
Talk about the LEE of O157
The LEE of O157 has an additional 7.5kb prophage sequence compared to its EPEC ancestor
The LEE is made of 41 different genes organised into three major regions:
- TTSS
- Intimin and TIR
- Esp
What is the TTSS of the LEE of O157?
A type III secretion system that exports effector molecules
Its a type 3 injector type mechanism
Bacteria needs contact with host for this mechanism to work
What is the intimin and TIR of O157?
Intimin is an adhesion
TIR is its translocated receptor which is translocated into the host cell membrane via the TTSS
What is the intimin and TIR of O157?
Intimin is an adhesion
TIR is its translocated receptor which is translocated into the host cell membrane via the TTSS
What is the Esp of the LEE of O157?
Several secreted proteins (Esp) as a part of the TTSS
Resopnsible for modification of host cel signal transsduction during the formation of A/E lesions
Explain in your own words how O157 attached to epithelium
Bacteria will bind to host cell initially with bundle forming pilus
Bacteia alter the cytoskeleton of host cell
A pedesal is formed
Host cell forms a receptor that the bacteria further bind to
Effacement will contibute
Give an indep description of how the LEE of O157 allows for binding of the bacteria to epithelium (video on brightsapce)
Talk about the plasmid of O157
Non-conjugative F-like plasmid a range size from 92 to 104 kb
pO157 shows a dynamic structure
mobile genetic elements such as transposons, prophages, insertion sequences (IS) and parts of other plasmids all together
19 genes found on the plasmid
What 19 genes are found on the plasmid of O157
a haemolysin (ehxA)
a catalase-peroxidase (katP)
a type II secretion system apparatus (etp)
a serine protease (espP)
a putative cytotoxin (toxB)
A zinc metalloprotease (stcE)
And an eae conserved fragment (ecf)
What is the haemolysin of O157 responsible for?
Haemorrhagic colitis
Talk about the carrying of shiga-toxin genes by phages
Stx-coding genes are carried by lambdoid prophages
Role of prophages is essential to microbial evolution
Increase evolutionary fitness
Rearrangement of genomic regions
Acquisition of foreign DNA
Spreading of phages has contributed to the emergence of different Shiga toxin producing E. Coli types
What are the effects of shiga toxins on cells in general
A-B toxins
They have a really potent cythopathic effect on many cell lines
Theyre particularly active on endothelial cells
Theyre lethal for laboratory animals
They inhibit protein synthesis
a = catalytic, biologicaly active part of toxin
How does shiga toxin affect the human host
Causes vascular damage mostly in the colon and kidneys
two major complications:
- haemorrhagic colitis (inflammation of the gut)
- haemolytic uremic syndrome
How does stx1 and xtx2 comapre t o each other
They both share the same enzymatic activity and structural features
However they are immunogically distinct
Stx2 is much more potent than stx1 in humans
Both are associated with the same haemorrhagic colitis and HUS
STEC strains encoding Stx2 are more likely to cause severe disease
What are the different parts of the shiga toxin
A divided into A1 and A2:
- Active site: glutamic acid
- Ribsome interaction region
- ER translocation region
B part is divided into 5 pentamiers
What is the mode of action of the shiga toxin - how is it activated?
A2 cleaved from A
The resultant A1 subunit is a highly specific N-glycosidase
This cleaves adenine 4324 from 28S rRBA comonent of eukaryotic 60S ribosomal subunit
Inhibiting elongation factor 1 (EF 1) dependent
Aminoacyl tRNA binding and peptide elongation
How does the toxin bind to and get into cells
Toxin (5b pentamers) binds to GB3 receptors expressed on endothelial cells in kidney and brain
Toxin GB3 complex is internalised by endocytosisi
Toxin-Gb3 complex undergoes retrograde transport through Golgi apparatus
Proteolysis leaves A1 and A2 subunits linked by a disulphide bond on the stem of a cleaved loop
In ER, the enzymatically active A1 fragment is liberated from the A2 fragment through disulphide-bond reduction
The A1 fragment cleaves an adenine in the 28S rRNA of 60S ribosomal subunits
This results in inhibition of protein synthesis and the induction of ribotoxic stress
How many subtypes and variants of stx are there?
There are 107 different subtypes and variants of these toxins
The stx2 toxins are associated with sever disease
How many subtypes and variants of stx2 are there?
7 subtypes (a-g)
35 variants
Talk about disease and certain subtypes of Stx2
Stx2 is more frequently associated with HUS and haemorrhagic colitis
Theres an association between HUS and the subtypes Stx2a, Stx2c and Stx2d
These trigger thombotic miroaniopathy (TMA)
What is Thrombotic microangiopathy
Thrombocytopenia < 150
Non immune haemolytic anaemia with a Hct <30%
Azotemia/high creatinine levels
How does shiga toxin affect endothelial cells
Enhanced expression of functional tissue factor that could contribute to microvascular thrombosis
Damage to or activation of endothelium, red cells and platelets
Damage to or activation of cellular responses such as cytokine expression and apoptosis caused by ribotoxic stress -> this then leads to inflammation along with infection
What are the main contributers to pathogenesis of O157
Phagosome island LEE
Shiga toxins
Plasmid O157
RpoS -> acid, heat and salt resistance
Iha -> adherence-conferring molecule
EAST1: enterotoxin
Various other LEE reulators and effectors
How are the effects of shiga toxin different in the brain
We see thrombotic effects to a lesser extent in the brain
How do the symptoms of O157 differ in terms of intestinal vs systemic infection
Intestinal:
- asymptomatic
- watery diarrhoea develops into HC
- haemorrhagic colitits
systemic:
- haemolytic uremic syndrome (HUS)
- microangiopathic haemolytic thromboytopenia
- anaemia
How does VTEC disease progress, how many recover vs die etcc
95% will recover but 5% will develop HUS
Out of this 5%:
- 3-5% will die
- 5% will siffer chronic renal failure, stroke and other major sequelae
- 30% will suffer proteinuria and other minor sequelae
-60% will resolve
Talk about the rate of HUS development from VTEC in different age groups
15.3% in those <5years old
7.9% in those 5-9 years
3.4% in those 10-17 years
1.2% in those 18-59
3.8% in those >60years old
NB: those 60+ had the highest rate of death due to VTEC whether or not they developed HUS
The frail and immunocompromised and pregnant are especially vulnerable
What are the HUS Risk Factors
Pathogen and host factors
Children most at isk
Leading cause of renal failure in children in the UK and USA
Some studies suggest female gender association
Association with Stx2a and severe disease
How is HUS treated
The use of antibiotics in the prevention of HUS area of intense speculation and debate
Plasma exchange to remove the toxin
Immunoadsorption
Future therapies such as shiga toxin binding or neutralisation therapies
Why is the use of antimicrobials for HUS debated?
As antimicrobials can stimulat the phages to produce more toxin
There have been outbreaks where they use antibiotics and the outcomes havent been any worse but its definitey not the recommended route
This is probably why we tend to see less resistance in STEC strains
Talk about research on EHEC non-O157 strains
There is relatively little known compared to O157
Limited knowledge as focus has been on O157 since 1982
Undestanding of ecology, reservoirs, transmission, virulence etc is all dependent on O157 research
How do the genes of O157 differ than non-O157
O157 possess classic EHEC genes such as:
- stx1/stx2
- LEE PAI
- pO157
- these maximise disease-causing potentia
Non-O157 are more viable:
- stx1 or stx2
-pO157 may be missing
- LEE PAI
Talk abou disease progression in non-O157 strains, who recovers who doesnt
Incubation of 3-4 days
Results in non-blood diarrhoea and abdominal cramps (60% recover at this point)
40% progress to bloody diarrhea after 1/2 days
98% resolve
Rare that HUS develops (less than 2%)
What is the ‘pathogenic paradigm shift’ in terms of E. coli
Germany outbreak marked the outing of E. Coli O104:H4 also known as enteroadherent haemorrhagic E. Coli (EAHEC)
This strain didnt have the classical virulence markers we would associate with normal EHEC but it was still extrardinarily virulent
What does EAHEC stand for?
Enteroadherent haemorrhagic E. Coli
Talk about the virulence factors of EAHEC
Lacked classical EHEC markers:
- LEE PAI
- pO157
Possessed:
- Stx2 (Stx2a)
- enteroaggregative E. Coli (EAEC) virulence factors
- aggregative adherence factor (AAF)
What was the EAEC outbreak?
Outbreak of a new strain in sprouts in germany in 2011
Toxin gene (stx2) was transferred via phage to an EAEC bacterium
New combination with additional resistance genes resulted in EAHEC O104:H4
Why is O104:H4 concidered a hybrid pathogen
It has the virulence of a VTEC STx2 strain as well as the adherence o an EHEC strain
Talk about adherence in O104:H4
AAF pilli encoded by aagR results in aggregation and adherence fimbriae
Stacked brick adherence
Thought to be down to unusual really tight binding on gut epithelium which allows for the quicker and increased delivery of toxins resulting in severe disease and a prolonged incubation period
Talk about the spread of EAHEC
Its an emerging pathogen but its spreading globally
It caused sporadic infections first in Asia and across Europe
Its endemic to central africa
What are the reservoirs for EAHEC
Not animals
Humans
Irrigation water contamination may be a source of EAHEC
What common EAEC virulence factors does O104:H4 also encode
AAF specifically AAF/I on pAA virulence plasmid
SPATE proteases
Dispersin aap gene
AggR global regulator
What additional virulence factors does O104:H4 encode that EHEC doesnt
Encodes a Stx2 within a lysogenize lambdoid bacteriophage
Plasmid that encodes an extended spectrum B-lactamase CTX-M-15
- Recent addition and not in the 2001 strain
Compare the disease progression of normal EHEC O157 vs STEC O1O4:H4
Only 5-10% of O157 develop HUS while 22% of O104 H4 will develop HUS
Why is there an increased risk of HUS with O104 than O157?
Aggregative adherence
Biofilm formation
Stx production
Prolonged release of toxin in this strain
How large was the initial O104:H4 outbreak?
Caused 4000 cases
Caused 53 deaths
Caused 855 cases of HUS
This was a really large outbreak, unusual, mortality and severe disease was high unlike other E. Coli strains
What did a retrospective study on the O104 outbreak reveal
Strain originated in sprouts
Tones of vegetables had to be dumped until the source was discoered -> was originally thought to be in cucumbers
Originated first in Egypt -> sprouts were only germinated in germany
Sprouts consumed in april, may and june
Outbreak began in may but was seen across all german hospitals -> sent to reference lab for toxn gvene detection and sequencing -> this was really the first time whole genome sequencing was done in real time, results ready in 4 days -> detected in leftover sprouts but never detected in original sporuts -> dont know how they got to sprouts etc
Talk about the epidemiology of the O104 outbreak
Young children who would normally be afected by E. Coli strains werent affected
Women were more commonly infected then males
- probably down to dietary choices and no other reason
Talk about epidemiology of STEC asociated HUS in Ireland (in general)
Trends in ireland generally mimic those described globally
90 cases between 2012 and 2014
83 culture positives
58% female
90% HUS<15
57% <5 years
3 HUHS cases in over 65s -> high mortality
What STEC serogroups in HUS and non-HUS are most common in Ireland
5 of the big 6 are seen (O26, O91, O145 O55, O103)
Rarer O91 serorgoup starting to see more of it in Ireland but it hadnt been one of the big six in the past
O157 = 50% of HUS cases
O25 = 33% of HUS cases
What toxin genotypes are seen in Ireland
4 stx 1s
33 stx 2s
15 stx 1 and 2
stx 2 has a higher risk of developing HUS (x4 times higher)
Any O157s had an stx2
Talk about the genetics of the HUS in Ireland
Predominance of Stx2 genotypes
13.5% were eae negative
67% were hylA negative
Small but important evolving STEC causing HUS
Any viable STEC has potential to cause HUS
Dont limit investigations in either clinical or food samples to merely the big 6 serogrous with eae positivity to define a virulent STEC strain
Why dont we use antibiotics for STEC
Ciprofloxacin signals SOS response
The phage will continuously be transcribed then resulting in continuous shiga toxin production
Hence why we dont treat with antibiotics just toxin neutralisers instead
Hence why we have little to no resistance
STEC strains are much slower to acquire resistance
Talk about resistance in O157:H7
Resistance to sulfonamide and tetracycline is common in O157:H7
There has been the detection of a shiga-toxin producing ESBL O157:H7 human clinical isolate in Denmark in 2013:
-blaTEM and blaCTX-M-1 blaTEM104 blaCTX-M-28
What resistance is seen in STEC O104
ESBL
Resistance to ampicillin, cefotaxime, ceftazidine, sulfamethoxazole, streptomycin, trimethoprim, tetracycline and naladixic acid
ESBLS have been seen in what other STEC strains other than O104
Three human isolates og O26 carrying either a blaCTX-M-3, blaCTX-M-18 or blaTEM-52
An O157 chicken isolate carrying a blaCTX-M-2 gene
