Enteric Bacteria + Viruses Flashcards
Describe how faeces samples are processed in MMUH
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
What would be the most probable organism in a liquid/bloody diarrhea sample?
Shigella or VTEC
What enteric organisms are notifiable diseases
All are notifiable
C. diff, Salmonella, Shigella, Campylobacter etc
For how long are samples kept
For 2 weeks
What must be done with any A&E samples?
Any liquid A&E samples must have a C.diff toxin test added on
Screen for C.diff
What should be done with a 3+ day old sample?
Sample should be rejected
When booking in any samples for C. diff testing what should you keep an eye out for?
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
How do we describe the appearance of samples?
Liquid
Soft
Semiliquid
Formed
Solid
What are the different tests that can be requested for enterics in MMUH
(5)
Gastro Panel 2 (Gastric panel)
C. Diff
Norovirus
Occult blood
Ovum parasites
What organisms are included in the Enteric Bio Gastro Panel 2?
Salmonella
Shigella
Campylobacter
Verotoxin producing E. Coli
Giardia
Cryptosporidium
How do we test for C. diff and norovirus?
Using Gene Xpert
Describe C. diff (4)
Clostridiodes Difficile
Anaerobic spore forming GPB
Farmyard Smell
Part of normal gut flora
Describe C. diff spores
Even though C. diff is an anaerobe the spores can survive in aerobic conditions
Spores can survive harsh environments
Spores can contaminate surfaces
How does C. diff cause infection
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
What antibiotic is most associated with C. diff infection
Clindamycin
How does Clindamycin cause C. diff infection?
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
How is C, diff transmitted
Often a hospital acquired infection
Spread through hygiene routes in healthcare setting e.g. hands of healthcare workers
What are the symptoms of C, diff
Liquid diarrhoea
Abdominal pain
Nausea
Vomiting
Loss of appetite
Fever
If you were to culture C. diff how would you do so
C. diff will grow on blood agar anaerobically
Can put up a metronidazole disc to prove anaerobic
What are some of the complications of C. diff
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
What are the virulence pathways of C. diff, how exactly does it cause symptoms etc
Toxin A or Toxin B producing C. diff attacks and invades enteric cells
Toxin A = enterotoxin
Toxin B = cytotoxin
How can you confirm ID of C. diff
Antigen test for glutamate dehydrogenase
Culture on anaerobic blood agar + metronidazole
Describe the use of antigen tests for C. diff
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
What are the genes of interest for C. diff and what do they encode
tcdA => toxin A => enterotoxin
tcdB => toxin B = cytotoxin
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
The C. diff genes tcdA and tcdB are part of what gene locus
PaLoc
What resistance is seen in C. Diff
Fluoroquinolone-resistance
How is C, diff genes detected
Using real-time PCR on the GeneXpert
Talk about Campylobacter
Microaerophilic GNB
S-shaped/Gullwing bacilli
Motile
Prone to drying out
Talk about Campylobacter infection
Most common cause of bacterial gastroenteritis
90% of Camp gastroenteritis caused by C. jejuni
Where is Campylobacter found?
Prefers having a host organism
Found in birds and stray dogs
How is Campylobacter spread?
Spread via contaminated food or water
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
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
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
How do we detect Campylobacter
Entericbio Multiplex PCR amplification and hybridisation using Gastro Panel 2 and Light Cycler 480
Detection of the HipO gene?
How can we confirm a Campylobacter?
CCDA put up with CampyGen sachet to provide ideal anaerobic conditions
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
Describe Salmonella
Flagellated NLF GNBs
Motile
Fimbriae
Capsulated
Two main species: S. enterica and S. bongari
Serovered into over 2400 serovers
Produce enterotoxins
Where is Salmonella found
Lives on the skin of lilzards or reptiles
Can contaminate food and water
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
How does Salmonella cause watery diarrheoa
Salmonella causes a huge sodium-chloride pump imbalance resulting in watery diarrhea leading to dehydration
What are the two main species of Salmonella
S. enterica
S. bongari
What Salmonella strain causes typhoid fever
S, typhi
How do we type Salmonella
Using the Kaufmann White Scheme
What is the Kaufmann White Scheme?
A method of typing Salmonella according to their combination of specific O and H antigens
What are the Salmonella antigens used in serotyping?
O antigen = somatic/cell wall antigen
H antigen = flagellar antigens
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
Where are any Salmonella positives send
National Salmonella and Shigella Reference Lab in Galway
What is the most frequent Salmonella strain we see?
S. typhi
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
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
What is DCA agar?
DCA agar (deoxycholate citrate agar) is a growth medium used for isolation and maximum recovery of Salmonella and Shigella
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
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
What information do we get back from the reference lab
Typing
Resistance report -> ID of any genes which confer resistance
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)
What are the four species of Shigella?
S. flexneri, S. boydii, S. sonnei, S. dysentriae
How does Shiga toxin work
Binds to enterocytes ribosomal units to disrupt protein synthesis
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
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
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
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
What do we do with the four slopes for Shigella
1 slope for serology
1 for reference lab
2 kept
Who is Shigella most common amogst?
Children, elderly
Childcare, nureseries, nursing homes etc
Gay and bisexual males
How is Shigella spread
Through faecal -> oral route
Through flies etc
What is the target gene for Shigella
Enteric Bio ID: ipaH = Shigella
BUT:
ipaH = Shigella + Enteroinvasive E. coli (EIEC))
stx1 = Shigella + STEC
What is the target gene for Salmonella
-> dont know
?PhoP gene
Describe VTEC
Verotoxigenic Escherichia coli
0157H7 is the most common E. Coli strain
Produces Shiga like toxin
What kind of infection does VTEC cause
Bloody diarrhea
haemorrhagic colitis and haemolytic uraemic syndrome (HUS)
Where is VTEC found and how is it spread
Associated with cattle
Spread through contaminated surfaces and food -> faecal/oral route
Can contaminate water
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
What would be a way of culturing VTEC
MacConkey agar
-> normal E. Coli = pink due to sorbitol fermentation
-> VTEC = colourless => non-sorbitol fermenter
Describe vibrio cholera
Flagellated comma shaped GNBs
Sensitive to stomach acid
Cholera toxin
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
Who might be more susceptible to Cholera
Those with improper acid secretion
Those on antacids
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
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
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
What does Salmonella vs Shigella look like on XLD?
Salmonella = black dot
Shigella = red colonies
What does Salmonella vs Shigella look like on DCA?
Salmonella = NLF w/black dot on DCA
Shigella = colourless
What would a yellow colony on XLD be?
A NLF
Probably just a Proteus or a Morganella
What is the most common Salmonella we get?
S. typhimurium
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
What is the target gene for Salmonella?
SpaO gene?
What is the most common Shigella
S. Sonnei
S. flexneri in MSMs
S. Boydi or dysentriae not found in Ireland
What is the target gene for Shigella
IpA gene
What should always be done before starting serology typing of organisms
Tests for auto-agglutination
How would you culture Campylobacter
CCDA agar @42 degrees
-> suppresses everything else
Microaerophilic conditions
How could you confirm ID of Campylobacter
MALDI
CCDA
Gram stain -> gullwing
Hippurate hydrolysis biochem test
Describe the use of hippurate hydrolysis for Campylobacter
Campylobacter jejuni (hip+) and Campylobacter coli (hip-)
What is the target gene for Campylobacter
tuf gene
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
- Hence why steak not a problem but mince meat is
How is VTEC treated and why such as way?
- Supportive therapy only
No antibiotics as this can often lead to system overload of toxins
What is the most common VTEC E.Coli
- O157 used to be the most common -> other newer types now more common
Talk about Vibrio
- Southeast asia
- Rice-water stool
- Never isolated in MMUH
- Seafood
- TCBS agar -> green agar
- Alkaline peptone water
Vibrio cholera = yellow
