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3.0 Microbiology > Enteric Bacteria + Viruses > Flashcards

Enteric Bacteria + Viruses Flashcards

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1
Q

Describe how faeces samples are processed in MMUH

A

All our testing is molecular based
We don’t initially put up plates for anything
We use PCR based methods through either the GeneXpert or the Enteric Bio + Roche Light Cycler
Plates only put up after IDing certain organisms such as Salmonella, Shigella, Cholera etc

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2
Q

What would be the most probable organism in a liquid/bloody diarrhea sample?

A

Shigella or VTEC

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3
Q

What enteric organisms are notifiable diseases

A

All are notifiable
C. diff, Salmonella, Shigella, Campylobacter etc

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4
Q

For how long are samples kept

A

For 2 weeks

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5
Q

What must be done with any A&E samples?

A

Any liquid A&E samples must have a C.diff toxin test added on
Screen for C.diff

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6
Q

What should be done with a 3+ day old sample?

A

Sample should be rejected

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7
Q

When booking in any samples for C. diff testing what should you keep an eye out for?

A

Appearance of sample -> can reject sample if not liquid
Check to see if there has been a previous positive C. diff within 4 week -> can reject sample if there is

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8
Q

How do we describe the appearance of samples?

A

Liquid
Soft
Semiliquid
Formed
Solid

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9
Q

What are the different tests that can be requested for enterics in MMUH
(5)

A

Gastro Panel 2 (Gastric panel)

C. Diff

Norovirus

Occult blood

Ovum parasites

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10
Q

What organisms are included in the Enteric Bio Gastro Panel 2?

A

Salmonella
Shigella
Campylobacter
Verotoxin producing E. Coli
Giardia
Cryptosporidium

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11
Q

How do we test for C. diff and norovirus?

A

Using Gene Xpert

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12
Q

Describe C. diff (4)

A

Clostridiodes Difficile
Anaerobic spore forming GPB
Farmyard Smell
Part of normal gut flora

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13
Q

Describe C. diff spores

A

Even though C. diff is an anaerobe the spores can survive in aerobic conditions
Spores can survive harsh environments
Spores can contaminate surfaces

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14
Q

How does C. diff cause infection

A

C. diff is part of normal gut flora in low numbers
C. diff infection is caused by disruption in gut microbiome resulting in overgrowth of C. diff
Microbiome disruption can be caused by any antibiotic but very high risk is associated with Clindamycin

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15
Q

What antibiotic is most associated with C. diff infection

A

Clindamycin

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16
Q

How does Clindamycin cause C. diff infection?

A

Clindamycin inhibits protein synthesis by attacking the 50s ribosomal subunit in bacteria which stops growth

Clindamycin will kill all other gut bacteria other than C. diff

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17
Q

How is C, diff transmitted

A

Often a hospital acquired infection
Spread through hygiene routes in healthcare setting e.g. hands of healthcare workers

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18
Q

What are the symptoms of C, diff

A

Liquid diarrhoea
Abdominal pain
Nausea
Vomiting
Loss of appetite
Fever

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19
Q

If you were to culture C. diff how would you do so

A

C. diff will grow on blood agar anaerobically
Can put up a metronidazole disc to prove anaerobic

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20
Q

What are some of the complications of C. diff

A

Toxic megacolon whereby the colon swells and may even burst to cause systemic infection

Pseudomembranous colitis -> due to prolonged inflammation + toxins + cytokines -> only found through colonoscopy and staining

Can cause systemic infection in the immunocompromised resulting in fever and hypotensive tachycardia

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21
Q

What are the virulence pathways of C. diff, how exactly does it cause symptoms etc

A

Toxin A or Toxin B producing C. diff attacks and invades enteric cells

Toxin A = enterotoxin
Toxin B = cytotoxin

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22
Q

How can you confirm ID of C. diff

A

Antigen test for glutamate dehydrogenase

Culture on anaerobic blood agar + metronidazole

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23
Q

Describe the use of antigen tests for C. diff

A

Some hospitals use a glutamate dehydrogenase antigen test to screen any samples for C. diff

Only antigen + samples will then go for GeneXpert to identify if toxin A or toxin B producing

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24
Q

What are the genes of interest for C. diff and what do they encode

A

tcdA => toxin A => enterotoxin

tcdB => toxin B = cytotoxin

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25
What do we do with positive C. diffs?
C. diff positives are frozen for ribocyclin -> whole genome sequencing -> this is to genetically type the C. diff for epidemiology trends and stats etc -> useful in tracing an outbreak etc Any positive patients are isolated from other patients -> put in a shared ward together
26
The C. diff genes tcdA and tcdB are part of what gene locus
PaLoc
27
What resistance is seen in C. Diff
Fluoroquinolone-resistance
28
How is C, diff genes detected
Using real-time PCR on the GeneXpert
29
Talk about Campylobacter
Microaerophilic GNB S-shaped/Gullwing bacilli Motile Prone to drying out
30
Talk about Campylobacter infection
Most common cause of bacterial gastroenteritis 90% of Camp gastroenteritis caused by C. jejuni
31
Where is Campylobacter found?
Prefers having a host organism Found in birds and stray dogs
32
How is Campylobacter spread?
Spread via contaminated food or water
33
What are the symptoms of Campylobacter?
Diarrhoea Abdominal pain Fever within 2-5days after ingestion Illness which lasts between 2-5 days or up to 10 days
34
How does Campylobacter cause infection?
Motile + S shape allows for penetration of gastrointestinal mucus Once adhered to enterocytes it destroys the lining of the gut by use of enterotoxins and cytotoxins
35
What are the affects of Campylobacter infection?
In 1/1000 infections it can cause Guillain-Barre syndrome through molecular mimicry of antigens present on the myelin sheath of the brain -> results in muscle weakness and even paralysis
36
How do we detect Campylobacter
Entericbio Multiplex PCR amplification and hybridisation using Gastro Panel 2 and Light Cycler 480 Detection of the HipO gene?
37
How can we confirm a Campylobacter?
CCDA put up with CampyGen sachet to provide ideal anaerobic conditions
38
What is CCDA and how does Campylobacter grow on it
Charcoal Cefoperozone Deoxycholate agar specific for Campylobacter Contains a cocktail of antibiotics which prevent the growth of other gut flora Camp grows as very small pinpoint grey colonies
39
Describe Salmonella
Flagellated NLF GNBs Motile Fimbriae Capsulated Two main species: S. enterica and S. bongari Serovered into over 2400 serovers Produce enterotoxins
40
Where is Salmonella found
Lives on the skin of lilzards or reptiles Can contaminate food and water
41
How does Salmonella cause infection
Invade enterocytes where they cause the release of cytokines Capsule causes fever Enterotoxin causes diarrhea VI capsule inhibits complement Salmonella invades and multiplies within enterocytes Causes pro-inflammatory release of cytokines -> this is what causes symptoms
42
How does Salmonella cause watery diarrheoa
Salmonella causes a huge sodium-chloride pump imbalance resulting in watery diarrhea leading to dehydration
43
What are the two main species of Salmonella
S. enterica S. bongari
44
What Salmonella strain causes typhoid fever
S, typhi
45
How do we type Salmonella
Using the Kaufmann White Scheme
46
What is the Kaufmann White Scheme?
A method of typing Salmonella according to their combination of specific O and H antigens
47
What are the Salmonella antigens used in serotyping?
O antigen = somatic/cell wall antigen H antigen = flagellar antigens
48
How do we serotype any positive Salmonellas?
Serotype using specific antisera We can only report the Salmonella group e.g. Group A Salmonella -> dont have the super specific antisera needed to further group the Salmonellas -> we then send them to the reference lab in Galway -> extended typing done here in NSSLRL
49
Where are any Salmonella positives send
National Salmonella and Shigella Reference Lab in Galway
50
What is the most frequent Salmonella strain we see?
S. typhi
51
What do we do with a positive Salmonella
Put up on XLD, DCA and Selenite broth (24hrs) Maldi XLD and DCA After 24hrs put up 2nd XLD using selenite broth Maldi 2nd XLD Send for Vitek Sensitivities and put up purity plate Send purity plate back to enterics for 6 slopes to be made
52
What is XLD agar?
Xylose Lysine Deoxycholate agar (XLD agar) is a selective growth medium used in the isolation of Salmonella and Shigella species Contains hydrogen sulfide which makes Salmonella colonies black coloured
53
What is DCA agar?
DCA agar (deoxycholate citrate agar) is a growth medium used for isolation and maximum recovery of Salmonella and Shigella
54
Why do we put up a selenite broth for Salmonella positives
▪ Inoculate selenite broth for 24hours before subculturing to an XLD ▪ Selenite helps any Salmonellas grow ▪ Particularly useful if nothing grows on the initial XLD or DCA Salmonella nearly always grows after time in the broth
55
What do we do with the six slopes put up for Salmonella positives
2 for serotyping using H and O antisera 1 for reference lab 3 kept incase reference lab sample not accepted etc
56
What information do we get back from the reference lab
Typing Resistance report -> ID of any genes which confer resistance
57
Describe Shigella
Non flagellated NLF GNB Causes shigellosis Produces shiga toxin Produces both exotoxins and endotoxins Causes acute watery/bloody diarrhea Four species: S. flexneri, S. boydii, S. sonnei, S. dysentriae Very low infectious dose (between 10 and 100 bacterial cells)
58
What are the four species of Shigella?
S. flexneri, S. boydii, S. sonnei, S. dysentriae
59
How does Shiga toxin work
Binds to enterocytes ribosomal units to disrupt protein synthesis
60
Talk about the Shigella toxins
Exotoxins -> enterotoxins + cytotoxin (Shiga toxin -> this is what causes cell rupture/bloody diarrhea)) Endotoxin -> released by auto-lysis LPS of bacteria also causes increased cytokines in infection etc
61
What are the affects of Shigella
Acute watery/bloody diarrhea which often causes dehydration HUS -> glomerular nephritis -> haemolytic uraemic syndrome -> shigella binds to epithelial cells of the kidney where it causes nephritis -> happens in 13% of cases
62
What do we do with any Shigella positives
DCA -> Shigella = orangey XLD -> Shigella = colourless Selenite broth (+ 2nd XLD) MALDI ID + VITEK ID + purity plate (sent back to enterics) 4 slopes from PP
63
What problems do we encounter when trying to ID Shigella
Enteric Bio may call a Shigella an EHEC (E. Coli) MALDI will confuse Shigella with E. Coli VITEK is the only way to get a positive Shigella ID -> will ID as 'Shigella group' Serology needed to type Shigella
64
What do we do with the four slopes for Shigella
1 slope for serology 1 for reference lab 2 kept
65
Who is Shigella most common amogst?
Children, elderly Childcare, nureseries, nursing homes etc Gay and bisexual males
66
How is Shigella spread
Through faecal -> oral route Through flies etc
67
What is the target gene for Shigella
Enteric Bio ID: ipaH = Shigella BUT: ipaH = Shigella + Enteroinvasive E. coli (EIEC)) stx1 = Shigella + STEC
68
What is the target gene for Salmonella
-> dont know ?PhoP gene
69
Describe VTEC
Verotoxigenic Escherichia coli 0157H7 is the most common E. Coli strain Produces Shiga like toxin
70
What kind of infection does VTEC cause
Bloody diarrhea haemorrhagic colitis and haemolytic uraemic syndrome (HUS)
71
Where is VTEC found and how is it spread
Associated with cattle Spread through contaminated surfaces and food -> faecal/oral route Can contaminate water
72
What do we do with VTEC+
Send straight to reference lab We don't culture it -> they will serotype the E. Coli -> they will tell us the exact gene producing the shiga-like toxin
73
What would be a way of culturing VTEC
MacConkey agar -> normal E. Coli = pink due to sorbitol fermentation -> VTEC = colourless => non-sorbitol fermenter
74
Describe vibrio cholera
Flagellated comma shaped GNBs Sensitive to stomach acid Cholera toxin
75
How does Cholera cause infection
Invades and multiplies in enterocytes Releases cholera toxin which binds to intestinal epithelial cells Causes a rise in cAMP which disturbs electrolyte balance: sodium:chloride -> watery diarrhea + dehydration Complications due to disturbances in acid:base balance
76
Who might be more susceptible to Cholera
Those with improper acid secretion Those on antacids
77
Talk about occult bloods
* Qualitative detection of blood in faeces * Card test -> qualitatively tells you if there is blood in stool or not -> not quantitative * Method: ○ Smear faeces onto reaction site on card ○ Fold card over and add reagent to back of card ○ Any positives will turn blue ○ Some samples may only turn blue around the edge where the sample is feathered out and thinner ○ Once card has three reactive sites, so for each sample it is done 3 times * You may be sent down samples from three consecutive days ○ Done to monitor treatment etc Not quantitative I.e. it doesn't tell you how much blood is present
78
Talk about norovirus
* Detected using the GeneXpert ○ Detects G1 and G2 genes * Causes symptoms such as diarrhea, abdominal cramping etc * Known as the 'winter bug' in children * Causes acute gastroenteritis *Non-enveloped single stranded RNA
79
Talk about the Enteric Bio
A workstation which does all the pipetting etc required for PCR Heating step of PCR must be done externally (make sure not to heat any one sample twice as this will cause damage to the single stranded DNA) Make sure not to overload when processing the sample as the PCR will fail
80
What does Salmonella vs Shigella look like on XLD?
Salmonella = black dot Shigella = red colonies
81
What does Salmonella vs Shigella look like on DCA?
Salmonella = NLF w/black dot on DCA Shigella = colourless
82
What would a yellow colony on XLD be?
A NLF Probably just a Proteus or a Morganella
83
What is the most common Salmonella we get?
S. typhimurium
84
Talk about Salmonella caused Enteric Fever
Seen in Typhi and paratyphi Salmonella invades enterocytes Spreads from enterocytes to bloodstream Causes enteric fever Will require antibiotic treatment
85
What is the target gene for Salmonella?
SpaO gene?
86
What is the most common Shigella
S. Sonnei S. flexneri in MSMs S. Boydi or dysentriae not found in Ireland
87
What is the target gene for Shigella
IpA gene
88
What should always be done before starting serology typing of organisms
Tests for auto-agglutination
89
How would you culture Campylobacter
CCDA agar @42 degrees -> suppresses everything else Microaerophilic conditions
90
How could you confirm ID of Campylobacter
MALDI CCDA Gram stain -> gullwing Hippurate hydrolysis biochem test
91
Describe the use of hippurate hydrolysis for Campylobacter
Campylobacter jejuni (hip+) and Campylobacter coli (hip-)
92
What is the target gene for Campylobacter
tuf gene
93
Where do we moat commonly seen VTEC contamination
* Mince meat * Abattoir -> perforated beast's bowel -> contaminate outside of cut of beef * Hence why steak not a problem but mince meat is Particularly a problem in summer when mince meat burgers arent cooked thoroughly enough
94
How is VTEC treated and why such as way?
* Supportive therapy only No antibiotics as this can often lead to system overload of toxins
95
What is the most common VTEC E.Coli
* O157 used to be the most common -> other newer types now more common
96
Talk about Vibrio
* Southeast asia * Rice-water stool * Never isolated in MMUH * Seafood * TCBS agar -> green agar * Alkaline peptone water Vibrio cholera = yellow
97

3.0 Microbiology (21 decks)

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