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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
Q

What do we do with positive C. diffs?

A

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

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26
Q

The C. diff genes tcdA and tcdB are part of what gene locus

A

PaLoc

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27
Q

What resistance is seen in C. Diff

A

Fluoroquinolone-resistance

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28
Q

How is C, diff genes detected

A

Using real-time PCR on the GeneXpert

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29
Q

Talk about Campylobacter

A

Microaerophilic GNB
S-shaped/Gullwing bacilli
Motile
Prone to drying out

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30
Q

Talk about Campylobacter infection

A

Most common cause of bacterial gastroenteritis
90% of Camp gastroenteritis caused by C. jejuni

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31
Q

Where is Campylobacter found?

A

Prefers having a host organism
Found in birds and stray dogs

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32
Q

How is Campylobacter spread?

A

Spread via contaminated food or water

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33
Q

What are the symptoms of Campylobacter?

A

Diarrhoea
Abdominal pain
Fever within 2-5days after ingestion
Illness which lasts between 2-5 days or up to 10 days

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34
Q

How does Campylobacter cause infection?

A

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

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35
Q

What are the affects of Campylobacter infection?

A

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

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36
Q

How do we detect Campylobacter

A

Entericbio Multiplex PCR amplification and hybridisation using Gastro Panel 2 and Light Cycler 480

Detection of the HipO gene?

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37
Q

How can we confirm a Campylobacter?

A

CCDA put up with CampyGen sachet to provide ideal anaerobic conditions

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38
Q

What is CCDA and how does Campylobacter grow on it

A

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

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39
Q

Describe Salmonella

A

Flagellated NLF GNBs
Motile
Fimbriae
Capsulated
Two main species: S. enterica and S. bongari
Serovered into over 2400 serovers
Produce enterotoxins

40
Q

Where is Salmonella found

A

Lives on the skin of lilzards or reptiles
Can contaminate food and water

41
Q

How does Salmonella cause infection

A

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
Q

How does Salmonella cause watery diarrheoa

A

Salmonella causes a huge sodium-chloride pump imbalance resulting in watery diarrhea leading to dehydration

43
Q

What are the two main species of Salmonella

A

S. enterica
S. bongari

44
Q

What Salmonella strain causes typhoid fever

A

S, typhi

45
Q

How do we type Salmonella

A

Using the Kaufmann White Scheme

46
Q

What is the Kaufmann White Scheme?

A

A method of typing Salmonella according to their combination of specific O and H antigens

47
Q

What are the Salmonella antigens used in serotyping?

A

O antigen = somatic/cell wall antigen
H antigen = flagellar antigens

48
Q

How do we serotype any positive Salmonellas?

A

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
Q

Where are any Salmonella positives send

A

National Salmonella and Shigella Reference Lab in Galway

50
Q

What is the most frequent Salmonella strain we see?

A

S. typhi

51
Q

What do we do with a positive Salmonella

A

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
Q

What is XLD agar?

A

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
Q

What is DCA agar?

A

DCA agar (deoxycholate citrate agar) is a growth medium used for isolation and maximum recovery of Salmonella and Shigella

54
Q

Why do we put up a selenite broth for Salmonella positives

A

▪ 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
Q

What do we do with the six slopes put up for Salmonella positives

A

2 for serotyping using H and O antisera
1 for reference lab
3 kept incase reference lab sample not accepted etc

56
Q

What information do we get back from the reference lab

A

Typing
Resistance report -> ID of any genes which confer resistance

57
Q

Describe Shigella

A

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
Q

What are the four species of Shigella?

A

S. flexneri, S. boydii, S. sonnei, S. dysentriae

59
Q

How does Shiga toxin work

A

Binds to enterocytes ribosomal units to disrupt protein synthesis

60
Q

Talk about the Shigella toxins

A

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
Q

What are the affects of Shigella

A

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
Q

What do we do with any Shigella positives

A

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
Q

What problems do we encounter when trying to ID Shigella

A

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
Q

What do we do with the four slopes for Shigella

A

1 slope for serology
1 for reference lab
2 kept

65
Q

Who is Shigella most common amogst?

A

Children, elderly

Childcare, nureseries, nursing homes etc

Gay and bisexual males

66
Q

How is Shigella spread

A

Through faecal -> oral route

Through flies etc

67
Q

What is the target gene for Shigella

A

Enteric Bio ID: ipaH = Shigella

BUT:
ipaH = Shigella + Enteroinvasive E. coli (EIEC))
stx1 = Shigella + STEC

68
Q

What is the target gene for Salmonella

A

-> dont know

?PhoP gene

69
Q

Describe VTEC

A

Verotoxigenic Escherichia coli
0157H7 is the most common E. Coli strain
Produces Shiga like toxin

70
Q

What kind of infection does VTEC cause

A

Bloody diarrhea
haemorrhagic colitis and haemolytic uraemic syndrome (HUS)

71
Q

Where is VTEC found and how is it spread

A

Associated with cattle
Spread through contaminated surfaces and food -> faecal/oral route
Can contaminate water

72
Q

What do we do with VTEC+

A

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
Q

What would be a way of culturing VTEC

A

MacConkey agar
-> normal E. Coli = pink due to sorbitol fermentation
-> VTEC = colourless => non-sorbitol fermenter

74
Q

Describe vibrio cholera

A

Flagellated comma shaped GNBs
Sensitive to stomach acid
Cholera toxin

75
Q

How does Cholera cause infection

A

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
Q

Who might be more susceptible to Cholera

A

Those with improper acid secretion
Those on antacids

77
Q

Talk about occult bloods

A
  • 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
Q

Talk about norovirus

A
  • 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
Q

Talk about the Enteric Bio

A

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
Q

What does Salmonella vs Shigella look like on XLD?

A

Salmonella = black dot
Shigella = red colonies

81
Q

What does Salmonella vs Shigella look like on DCA?

A

Salmonella = NLF w/black dot on DCA
Shigella = colourless

82
Q

What would a yellow colony on XLD be?

A

A NLF
Probably just a Proteus or a Morganella

83
Q

What is the most common Salmonella we get?

A

S. typhimurium

84
Q

Talk about Salmonella caused Enteric Fever

A

Seen in Typhi and paratyphi
Salmonella invades enterocytes
Spreads from enterocytes to bloodstream
Causes enteric fever
Will require antibiotic treatment

85
Q

What is the target gene for Salmonella?

A

SpaO gene?

86
Q

What is the most common Shigella

A

S. Sonnei

S. flexneri in MSMs

S. Boydi or dysentriae not found in Ireland

87
Q

What is the target gene for Shigella

A

IpA gene

88
Q

What should always be done before starting serology typing of organisms

A

Tests for auto-agglutination

89
Q

How would you culture Campylobacter

A

CCDA agar @42 degrees
-> suppresses everything else

Microaerophilic conditions

90
Q

How could you confirm ID of Campylobacter

A

MALDI
CCDA
Gram stain -> gullwing
Hippurate hydrolysis biochem test

91
Q

Describe the use of hippurate hydrolysis for Campylobacter

A

Campylobacter jejuni (hip+) and Campylobacter coli (hip-)

92
Q

What is the target gene for Campylobacter

A

tuf gene

93
Q

Where do we moat commonly seen VTEC contamination

A
  • 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
Q

How is VTEC treated and why such as way?

A
  • Supportive therapy only
    No antibiotics as this can often lead to system overload of toxins
95
Q

What is the most common VTEC E.Coli

A
  • O157 used to be the most common -> other newer types now more common
96
Q

Talk about Vibrio

A
  • Southeast asia
  • Rice-water stool
  • Never isolated in MMUH
  • Seafood
  • TCBS agar -> green agar
  • Alkaline peptone water
    Vibrio cholera = yellow
97
Q
A

3.0 Microbiology (21 decks)

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