STEC Flashcards

1
Q

List some of the different variants of E. Coli

A

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)

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2
Q

EPEC, ETEC and DAEC cause infections where in the body?

A

Colonise the small bowel and cause diarrhoea

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3
Q

EHEC and EIEC cause infections where?

A

They cause disease in the large bowel

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4
Q

EAEC infects what part of the body?

A

Can colonise both the small and large bowels

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5
Q

Where does UPEC cause infection

A

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

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6
Q

Where does NMEC cause infection

A

It can cause septicaemia

Neonatal meningitis E. Coli can cross the blood brain barrier into the central nervous system and cause meningitis

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7
Q

What E. Coli strains colonise the small bowel and cause diarrhoea

A

EPEC
ETEC
DAEC

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8
Q

What E. Coli strains cause disease in the large bowel

A

EHEC
EIEC

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9
Q

What E. Coli strains colonoise both the small and large bowel

A

EAEC

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10
Q

What E. Coli strains cause septicaemia

A

UPEC
NMEC

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11
Q

Who are most at risk of urosepsis

A

Elderly women -> UTIs progress into septicaemia

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12
Q

How do the E.coli strains differ from one another

A

They differ in terms of pathogenicity

some inherit toxins such as shiga toxin in EAHEC

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13
Q

What is VTEC

A

Verotoxigenic E. Coli

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14
Q

What are the genes for VTEC, what do they encode

A

Stx1 and Stx2

Verotoxins encoded on bacteriophages

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15
Q

What are the genes for EHEC and what is their function

A

vtx1, vtx2, eae genes

Haemorrhagic colitis hence enterohaemorrhagic E. Coli

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16
Q

How was E. Coli traditionally serotyped?

A

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

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17
Q

Give some examples of E. Coli strains named according to O, H and K antigens

A

O157:H7
O104:H4
O26
O103
O111

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18
Q

Where did verotoxigenic E. Coli get its name

A

VTEC comes from the old assay that was used to identify verotoxin producing e. coli

Vero monkey cells

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19
Q

What two organisms produce shiga toxin

A

Shigella dynsentriae type 1
Shiga toxin-producing E. Coli

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20
Q

Traditionally how was functionally active shiga toxins detected?

A

Using vero cell toxicity test

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21
Q

What strains will be vero cell toxicity test positive?

A

Verotoxin or verocytotoxin-producing E. Coli (VTEC)

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22
Q

What does shiga toxin cause?

A

Diarrhoea
Haemorrhagic colitis
Haemolytic uremic syndrome (HUS)
Enterohaemorrhagic E. Coli (EHES)

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23
Q

Technically speaking Shiga toxin could be produced by any E. Coli why is this?

A

Since the gene for it is transferred via bacteriophage so technically any strain can take it up

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24
Q

Talk about STEC/Shiga toxin E. Colli, what is it, how many different types are there

A

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

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25
Is stx1 or stx2 more toxigenic
Stx2 is 1000 times more toxic
26
What is the best studied strain of STEC
E. Coli O157:H7
27
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
28
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
29
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
30
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
31
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
32
What were the three main O157:H7 outbreaks studied in America
Spinach (SP) Taco Bell (TB) Taco John (TJ)
33
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
34
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
35
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%)
36
How many known differnet SNP genotypes are there for O157:H7?
39 SNP Genotypes 20% SNP difference at 96 loci 9 distinct clades
37
What is clade 8 of O157:H7 associated with?
Hypervirulence but also clinical illness and more frequently reported blood diarrhoea
38
What are clades 2, 7, and 8 of O157:H7 associated with?
These are the clades associated with clinical illness
39
What are clades 2 and 8 of O157 associated with?
Clades 2 and 8 are associated with increased reports of bloody diarrhoea
40
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
41
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
42
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
43
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
44
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
45
Which of the non-O157 strains are most common
O26 O111 O103 O145 In this order -> O26 and O111 similar numbers
46
What factors contribute to the virulence of O157:H7
adhesion LEE Haemolysin (encoded by plasmid) ToxB -> prophage Mobile genetic elements: - phages - genomic islands - plasmids
47
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
48
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
49
Need detailed descripion of tight binding of O157:H7 to epithelium
50
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)
51
What is eae
attaching and effacing lesion Its a gene that encodes intimin Its responsible for the effacement of microvilli in EHEC
52
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
53
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
54
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
55
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
56
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
57
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
58
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
59
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
60
Give an indep description of how the LEE of O157 allows for binding of the bacteria to epithelium (video on brightsapce)
61
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
62
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)
63
What is the haemolysin of O157 responsible for?
Haemorrhagic colitis
64
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
65
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
66
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
67
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
68
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
69
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
70
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
71
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
72
How many subtypes and variants of stx2 are there?
7 subtypes (a-g) 35 variants
73
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)
74
What is Thrombotic microangiopathy
Thrombocytopenia < 150 Non immune haemolytic anaemia with a Hct <30% Azotemia/high creatinine levels
75
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
76
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
77
How are the effects of shiga toxin different in the brain
We see thrombotic effects to a lesser extent in the brain
78
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
79
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
80
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
81
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
82
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
83
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
84
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
85
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
86
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%)
87
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
88
What does EAHEC stand for?
Enteroadherent haemorrhagic E. Coli
89
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)
90
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
91
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
92
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
93
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
94
What are the reservoirs for EAHEC
Not animals Humans Irrigation water contamination may be a source of EAHEC
95
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
96
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
97
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
98
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
99
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
100
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
101
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
102
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
103
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
104
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
105
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
106
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
107
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
108
What resistance is seen in STEC O104
ESBL Resistance to ampicillin, cefotaxime, ceftazidine, sulfamethoxazole, streptomycin, trimethoprim, tetracycline and naladixic acid
109
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