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Clinical Micro > Lecture 9 C. difficile > Flashcards

Lecture 9 C. difficile Flashcards

1
Q

What is CDAD

A

Clostridium difficile associated disease

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2
Q

What is the hypervirulent and hypertoxic strain from canada

A

Ribotype 027

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3
Q

How can a c diff infection be characterised

A

Faecal-oral transmission
Food associated infection
Food-poisoning
Antibiotic associated diarrhoea

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4
Q

What is the difference between food associated infection and food poisoning

A

Food associated infection is where food acts as vehicle for transmission, so food handlers contaminate the food and then the pathogen is consumed eg. Salmonella sp. , camplyobacter jejuni, ecoli 0157

Food poisoning is where the toxin is present in the food so the toxin (not pathogen) is consumed e.g. Staph aureus

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5
Q

Where is faecal-oral transmission common and whic microbes cause it `

A

Institutions such as nurseries, with contaminated hands/fomites and food.

Rotavirus, norovirus

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6
Q

How does AAD lead to GI

A

beta lactams and broad spectrum antibiotics give people diarrhoea so gut become ‘sterile’ lose normal flora so pathogen bacteria overgrowth

E.g. CLOSTRIDIUM DIFFICILE

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7
Q

When was c diff first discovered and what was it named

A

Bacillus difficilis as hard to grow in 1935

Normal component of faecal flora of 70% newborn babies and 2-3% of healthy adults.

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8
Q

Name some characteristics of morphology and physiology of.c diff

A 
Large GPR 15-20 microns in length
Strictly anaerobic 
Spore forming organism (ubiquitous) 
Toxin producing
>100 different genetic types (ribotypes) 
Ribotypes 001, 106 and 027 common in UK
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9
Q

Which ribotypes of c diff are common in uk

A

Ribotypes 001, 106 and 027

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10
Q

What do strictly anaerobic microorganisms use as terminal electron acceptor in ETC

A

Reduce sulphites instead of oxygen, which are less oxidising . Therefore organism produce less ATP and take longer to grow (approx 2 days)

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11
Q

C difficile is one of the leading HAI in the UK associated with AAD, pseudomembranous colitis and death. true or false

A

True

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12
Q

What was the approximate number of cases of c diff in hospitals in uk in 2005

A

46,000

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13
Q

What year did ribotype 027 hit uk

A

2004

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14
Q

Describe two campaigns introduced (w/ dates) and describe the trend

A

55,000 cases in 2006
Introduce clean your hands campaign around 2004 - break chain of infection.

2008ish - the deep clean - matrons brought back to wards and responsible for intensive chlorine based disinfection s followed by hydrogen peroxide vapour so can get into nooks and cranny in the ward to get spores.

After this see cases drop to approx 12,000 in 2012

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15
Q

In 2008, _% of death certificates stated c diff was the underlying cause

A

50%

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16
Q

When was the peak year of 027 strain in hospitals

A

2007

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17
Q

What are the risk factors for c diff

A 
As spores are transmissible, resistant to antimicrobials and persistent, these are at risk:
Hospitalisation 
Age >65
Broad spectrum antibiotic use 
Hygiene/dirty environment 
Close proximity of patients 
Nasogastric tubes
Contaminated equipment e.g blood pressure cuffs.
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18
Q

C. difficile is a commensal, true or false

A

True

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19
Q

What % of blood pressure cuffs are contaminated with spores and why

A

33% due to velcro, hard to clean

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20
Q

Describe the structure of c diff spores

A

Proteinacious coat on outside, peptidoglycan cortex in the middle and the coats/cortex protect the core which contains the bacterial DNA.

Also in the spore is dipicolinic acid and calcium, and SASPS (small acid soluble spore proteins) that surround DNA and protect it from environmental extreme.

Only 30% water so can live in dehydrated environment

21
Q

Summarise the infection pathway

A

1- susceptible patient ingests spores which resist stomach acid
2- small intestine favourable conditions for c difficile spore germination: pH 6-6.8, bile salts (SODIUM TAUROCHOLATE), amino acids (glycine, histidine, aspartic acid, arginine and valine) which a co-germinants with sodium taurocholate
3- vegetative cells: flagellated, metabolically active produce potent toxins once adhered to small intestine. Produce symptoms such as diarrhoea and possibly PMC
4- Vegetative cells produce spores in the colon as unfavourable conditions and get excreted in patient diarrhoea as spores which can contaminate further

22
Q

Describe the germination of C. difficile spores in the small intestine

A

Germination intimation : germinants: sugars, aa, nutrients, bile salts
Loss of heat resistance and ions (K+, H+ and Na+) and Ca-DPA complex so get
- partial rehydration of core
- hydrolysis of cortex and degradation of SASPs
-Rehdyration of core and restoration of metabolic activity
- sensitive to antimicrobials

23
Q

How quickly can a dormant spore become metabolically active

A

Within 20 minutes

24
Q

What adherence virulence factors does c diff have

A

S layer proteins (adhesins) and peritrichous flagella (secondary attachment)

25
Q

What toxins are produced by c diff

A 
Genes on PaLoc
toxin A (enterotoxin) 
Toxin B (cytotoxic)
26
Q

What does toxin A (TcdA) and ToxinB (TcdA) do

A

Induce glycosylation of G proteins (Rho, Rac, cdc42) and affect actin polymerisation and therefore loss of tissue integrity

Both toxins induce an inflammatory response involving IL-8, Il6, TNFa, leptin and substance P.

27
Q

What receptor does TcdA stimulate

A

Vanilloid receptor VR1 which leads to release of substance P from sensory neurones (and increases expression of its receptor neurokinin 1-R), which in turn leads to release of neurotensin and mast cell degranulation

28
Q

TcdA also increases the release of _ which increases the release of more substance _ via stimulation of _

A

Endocannabinoids
P
VR1

29
Q

When both toxins induce IL8 secretion from sensory neurones and colonocytes, what cells are attracted to this.

A

Infiltration of polymorphonuclear leukocytes (PMNs) into the lamina propria

30
Q

Describe the clinical spectrum of c diff infection

A

Asymptomatic (25% of strains non toxin producing)
Explosive diarrhoea (release of organisms - vegetative and spores. If not treated properly ->
Pseudomembranous colitis (PMC) if not treated properly ->
Fulminant colitis (3%)
Can get rupture and organisms can enter blood stream - may die if not treated properly (approx 1700 uK)
Death

31
Q

What is differnt about ribotype 027

A

Produce more toxin A and B
Base pair deletion in toxin regulating gene
More resistant to ciprofloxacin and moxifloxacin (quinolones) than other c diff

32
Q

How many infections and deaths were at Maidstone & tunbridge wells hospital in 2006

A

90 deaths, 1170 infections

33
Q

Describe PaLoc structure and function with reference to 027 mutation

A

TcdD first - positive regulator, switch on toxin production in stationary phase of growth
TcdB - encode toxin B
TcdA encode toxin A
TcdC - negative regulation to stop toxin production - base pair deletion here in 027. Single bp deletion at position 117, 18 bp deletion at 330-347.
TcdE encode for porin in bacterial cell which allow toxin to be released

34
Q

Why is the deletions in tcdC detrimental to patient AND BACTERIA

A

Patient high levels of production

Bug - need lots of energy to produce these toxins

35
Q

Both tcdB and TcdA contain 3 domains, what are they and what are their function (extra)

A

Domain E (glucosyltransferase): active enzyme domain.
Domain B responsible for binding to host receptors which consists of multiple oligopeptide repeat units
Domain T - involved in translocation of toxin across host cell membrane

36
Q

What poor conditions did the investigation into maidstone and tunbridge wells find

A

Close patient contact
Filthy conditions: shower cubicles, staff eating and drinking areas, faecal stained commodes

SPORES THRIVE HERE

37
Q

How is c diff diagnosed in the lab

A 
Clinical sample (diarrhoea) 
Cat 2 pathogen

Non culture techniques - toxin detection (cell lines/EIA), glutamate dehydrogenase (GDH) detection (EIA) as enzyme is unique to c diff in clostridium genus
Culture - CCFA selective media

Typing : level 4 identification - ribotyping in outbreak situation

38
Q

Describe the toxin detection methodology of cell lines

A

Use HeLa and Hep2 to grow confluent in flasks. Add suprnatent of spun faeces which contains toxin. If present get cytopathic effect and see cell rounding.
To confirm it is c diff, neutra;use toxin with C.sordelli anti toxin and reinoculate fresh cellline and wouldnt get rounding

Take approx 1-3 days.
Sensitivity 94-100%/ specificity 99%

39
Q

Describe the other toxin detection method, EIA (widely used)

A

Lateral flow method. Detect toxins and gluamate dehydrogenase. Add faecal sample to EIA - look like pregnancy test with Line + dots = positive sample, Lines both sides of dots = c diff is present and producing toxin and dots only = negative for antigen and toxin.

Sensitivity 71-94% not as good but rapid result

40
Q

What conditions is CULTURE of sample used for c diff

A

Outbreak situations , otherwise use non culture

41
Q

What media is used and what do colonies look like (plus identification)

A

CCFA (cefoxitin, cycloserine, fructose) - selective to get rid of normal faecal flora.

Ground glass medusa head colonies, with odour of horse manure.

Can identify bacteria using API rapid ID 32A or Maldi TOF

42
Q

When would ribotyping be performed

A

Outbreak situation

43
Q

Why is ribotyping used for c diff

A

Germanic DNA encoding for 16S and 23S rRNA is HIGHLY CONSERVED region e.g. Genetic info coding for rRNA vary less within bacteria of same strain than differnt strains - HIGHLY DISCRIMINATORY

44
Q

What is the basic principle of ribotyping for c diff

A

Genomic dna digesting with restriction endonucleases and ran on gel electrophoresis
Specific universal oligonucleotide probes target conserved regions 16S and 23S rRNA coding region

Visualised using ethidium bromide or equivalent
Band pattern compared against database of established c diff ribotypes

45
Q

How many clostridium difficile ribotyping network (CDRN) service labs are there in uK

A

9 , one in heartlands

46
Q

What are the treatment strategies for c diff infection

A

Discontinue antibiotic causing it
Replace fluid and electrolytes
Antibiotics for treating c diff - oral vancomycin (250mg ; 10-14 days) act on cell wall, oral metronidazole (250mg 10-14days) (act on bacterial DNA so good for anaerobic)

Biotherapy restore gut microbiome, give probiotics such as saccharomyces boulardii rapidly recolonise gut.S boulardii also produce protease that splits c diff toxin A so double approach

Biotherapy of faecal transplantation - take faeces from donor and place it in patient gut after screening. Supernatant of spun faeces is given to patient to small intestine by nasogastric tube, or can be in faecal enema

47
Q

What other therapies are available except bio therapy and antimicrobial

A

Cholestyramine - bind toxin but also vancomycin so cant give same time

Monoclonal anti-toxin antibodies

Intravenous immunoglobulin? Not clear yet

48
Q

How can c diff be [prevented

A

Strict control on antibiotic use - narrow spectrum (get antibiotic sensitivity pattern first!)

Prompt implementation of precautions - isolation of patients into cohort wards. Effective hand hygiene. Disposable equipment e.g. Single use rectal thermometers

Eliminate spores from the environment - spores resistant to common disinfectants , need to use nasty chemicals at the moment to get rid such as aldehydes and superoxide. Soap and detergent as physical removal will help in combination with other measures.
Including germinate to exterminate solution - wake cells from spores to germinate cells (using a germination solution with antimicrobial) so become sensitive to antimicrobials and then are killed

Clinical Micro (9 decks)

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