clostidioides difficile

Question 1

the organism

Answer 1

• gram-positive, obligate anaerobe, rod-shaped
• forms spores - highly resistant to heat and the chemicals
• produces exotoxins
• spectrum of disease from diarrhoea to pseudomembranous colitis
• extra-intestinal manifestations rare
• important nosocomial infection

Question 2

role of antibiotics in C.difficile infection

Answer 2

• pseudomembraneous colitis was rare until 1970s when hospital outbreaks were first observed
• due to use of broad spectrum antibiotics killing colonic bacteria
• allowed over growth of C.dificile that may have been present in low numbers
• most common healthcare associated infection
• in high income countries the most common cause of infectious diarrhoea in hospitals
• up to 35% of cases result in recurrent infection

Question 3

spectrum of disease caused by C.dificile

Answer 3

• transient infection with clearance
• asymptomatic colonisation
• mild diarrhoea
• more severe disease syndromes include abdominal pain, fever and leukocytes
• severe infection is characterised by inflammatory lesions, formation of pseudomembranes in the colon, toxic megacolon, bowel perforation, sepsis, shock and death
• outcome depends on both strain properties and host immune response

Question 4

diagnosis

Answer 4

• foul-smelling diarrhoea is indicative of infection
• gold standard is a culture of organisms on selective media with an anaerobic environment followed by the detection of toxin through a cell toxicity assay
• key consideration is that culture alone cannot differentiate asymptomatic colonisation from infection so need to know if strains are producing toxins

Question 5

how many cases

Answer 5

estimated 450,000 C.dificile infections and 15,000 deaths annually in the US

Question 6

2005 - global epidemic

Answer 6

in 2005 - C.dificile strain with increased severity, transmission and mortality emerged in North America
- known as BI/NAP/027
- group BI by restriction endonuclease analysis
- in addition to TcdAB toxins this strain also produces binary toxin also referred to as C.dificile transferase

Question 7

treatment options for symptomatic C.dificile infection

Answer 7

• stop treatment with precipitating antibiotics
• new antibiotic regimen:
  
  • metronidazole - milder cases
  • vancomycin - more severe
  • key issue is that these antibiotics still adversely affect the gut microbiota leading to recurrences.

Question 8

antibiotic treatment - fidaxomicin

Answer 8

• new antibiotic treatment option (approved 2011) for severe cases with a narrower spectrum of activity and reduced recurrence of infection but very expensive

Question 9

faecal microbiota transfer

Answer 9

replenishment of flora through faecal transplant via enema/ nasogastric tube
- cure rate of over 90%
- restored diversity of gut microbiota

Question 10

stool banks for faecal microbiota transfer

Answer 10

faeces blended in saline, sieved and centrifuged to concentrate.
glycerol added as a cryoprotectant for long term cold storage in capsules at -80 degrees

Question 11

improvements of the FMT approach

Answer 11

• major issue with FMT is the use of undefined materials with potentially unintended consequences
• next challenge is to identify which species within the faecal microbiota can protect against C.dificile infection
• key paper identifies bacterial species that are associated with protection against C.dificile
• uses a subset of these to show protection in a mouse model of C.dificile infection
• protection was associated with the ability of these species to produce secondary bile acids that C.dificle is sensitive to

Question 12

key virulence factors - cytotoxins

Answer 12

TcdA and TcdB - the major virulence factors:
- causes secretory diarrhoea and colonic mucosal inflammation
- disrupt actin cytoskeleton and tight junction
- decreased transepithelial resistance, fluid accumulation and destruction of epithelium

C.difficile transferase toxin
- Also known as binary toxin, unrelated to TcdA/B
- role in disease not well understood

Question 13

key virulence factors - surface layer proteins

Answer 13

array of often glycosylated proteins on the cell surface

Question 14

key virulence factors - sporulation

Answer 14

• key to spread within hospital environment
• resistant to desiccation, chemicals and high temperature

Question 15

cytotoxins TcdA and TcdB

Answer 15

• belong to large clostridial toxin family
• single chain proteins composed of four functional domains:
• amino terminal catalytic domain with glucosyltransferase activity
• auto protease domain
• delivery domain for receptor binding, pore formation and toxin translocation
• carboxy terminal combined repetitive oligopeptide sequences domain
• fuunction to glycosylate small GTPases (Rho/Ras families):
• render inactive leading to cytoskeletal changes within host cell

Question 16

TcdAB mediated intoxication of host cells

Answer 16

-TcdAB toxins bind diverse receptors
- TcdA and TcdB are taken up by a clathrin-independent and clathrin-mediated endocytosis respectively
- the intravacuolar pH lowers due to proton influx causing conformational change in TcdAB and pore formation
- APD and GTD translocate into the cytosol and APD is activated by inositol hexakisphosphate binding causing autoproteolytic mediated release of GTS
- host GTPases are monoglucosylated by GTD

Question 17

overview of C.dificle pathogenesis - step 1

Answer 17

following disruption of the intestinal flora, C.difficile colonises
- if a toxigenic strain then TcdAB are produced, bind to the apical cell surface, are internalised inducing cytoskeletal changes including tight junction disruption and subsequent breakdown
- the subsequent release of inflammatory mediators attracts neutrophils

Question 18

overview of C.dificle pathogenesis - step 2

Answer 18

with epithelial barrier breakdown C.difficile toxins cross the epithelium with TcdB binding preferentially to the basolateral cell surface