C. difficile Flashcards
What kind of bacteria is it
Gram positive, spore forming, anaerobic bacterium (completely areo-intolerant)
Its an ancient firmicute
What does C. difficile cause
Causes spectrum of disease - collectively known as CDAD (C. difficile associated disease)
What are symptoms of CDI
Diarrhoea (which is usually self limiting and doesn’t require intervention)
In rare cases, this can be recurring and often gets worse with each sequential infection
Death from C. difficile is most commonly from Pseudomembranous colitis (blisters filled with neutrophils that infiltrate the tissue of the large intestine leading to perforation of the large bowel and causing sepsis)
and toxic megacolon (intestines become so swollen, they pop out of the abdomen, surgery is the only treatment and patients lose their entire colon
Deaths in the UK - 2000
What is C. difficile the cause of and where is most commonly seen
Hospital acquired infection worldwide (10c more common in hospitals than S. aureus)
Most commonly seen in the elderly but increasingly seen in the community and younger populations (children and pregnant women)
Also seen in animals (pigs, cattle, horses, chickens)
What are the associated healthcare costs
Associated healthcare costs
United States – $3.2 billion/year
Europe – €3 billion/year- just tackling c. difficile infection
Hard to disinfect hospital wards
Whats the disease progression
- Patient receives antibiotics
- alterations in the normal gut flora (allows C> diff to proliferate)
- Infection with C. difficile spores (metabolically inert spore)
- Spores germinate in gut forming vegetative cells
- Cells multiply, produce toxins and sporulate (helps them survive in the environment, leading to the spores being excreted
What are the toxins and what do they cause
TcdA and TcdB which cause diarrhoea and allows them to spread back into the environment
How long does the germination process take
Around 90 minutes (rapid process)
timeline of C. difficile
1935: Isolated from human infant gut and was named Bacillus difficile
1978: Established as a major cause of antibiotic associated diarrhoea (through the application of kochs postulates
1980-2000: Recognised as a gut commensal bacterium in ~ 5% healthy adults- lot of the time, its commensal. Toxins A and B characterised as major virulence factors. Mode of action of toxins established early on
2003: Epidemics in Quebec and Philadelphia; attributed to new fluoroquinolone resistant ”hypervirulent strains” (NAP1/027)
2004-2005: Epidemic strains appear in UK, and in several other European countries
2008: Other hypervirulent strains (078) appear in hospitals; such strains previously associated with animal infection- not associated with human infection - zoonotic strain
2016: Renamed Clostridioides difficile to reflect new understanding of the scale of genomic variation in the wider Clostridia family
C. difficile related deaths in England and Wales
Increased number of deaths in 2007 (8000). Then went back down to 2000 deaths in 2012.
The outbreak was associated with the introduction from a new hypervirulent strain from North America
They tackled this by using soap and water to it gets rid of spores.
Some countries in Europe haven’t got this outbreak under control
C. difficile ribotypes in England
The ribotypes in the UK are variable and there is little evidence of hospital transmission and 027 became under control
North American Outbreak - “Hyper-virulent” strains
Georgia, Pennsylvania, Maine, New Jersey, Oregon and Pennsylvania 2000-2003
Quebec 2003
Strain:
NAP1 (North American Pulse-field electrophoresis Type 1), Ribotype 027: how they discriminating the strains
Fluoroquinolone resistant
1 outbreak??
Ribotype 027 was first isolated in 1984, but what had changed???
What were the hypervirulent strains
027/ NAP1 strains are not new but:
Previously rare
Now fluoroquinolone resistant (suggests recent acquisition!)
Associated with large outbreaks – more transmissible/ more successful for going to patient to patient?
Associated with more severe disease – hyper-virulent?
Characteristics:
Produces more spores- accounts for them being more transmissible, more infectious to patients around them
Produces more toxin in vitro
Produces a third toxin – binary toxin (CDT)
Contain a 18 bp deletion in tcdC (anti-Sigma factor- negatively regulates expression of toxins)
Canadian and USA strains arose from ancestral strain independently – separate acquisition of fluoroquinolone resistance
What does C. difficile eat
Increase in Trehalose after 2000
C. difficile 027 became good at eating trehalose
Epidemic ribotype 027 has a point mutation in treR -> >500 fold more sensitive to trehalose (reacts really well to even low concentrations in the environment- good at scavenging trehalose)
Deletion of treA attenuates Ribotype 027, supplementation with trehalose enhances virulence
Ribotype 078 has a new 4 gene trehalose transport and degradation operon
How does the microbiome defend against C. diff
Bacteria outcompeting c. difficile for food
Host metabolites modulated by bacteria in gut
Bacteria can produce bacteriocins which kill other bacteria
Bacteria modulating host immune response (c. difficile is sensitive too)- good at preventing pathogens getting in
Pathogens have to get past the natural ecosystem of the human microbiome- features are true for most pathogens
What is the prerequisite for CDI
Dysbiosis
Microbiota recovers as infection is cleared
How can we exploit the microbiota to treat infection
Faecal microbiota transplantation
Spores are:
- Highly infectious
- Resistant to various chemical and physical insults
- Persistent in health care facilities for long periods of time
How are spores so resistant
- Down to the organisation of the structure:
Highly cross linked protein coats on the outside of the spore: self assembling proteins that assemble into 2D crystals (striations on the outside of the spore) and further chemically cross linked. They cannot be removed easily and they’re so strongly linked to each other they will not come off.
Cortex: massive thick cell wall/ peptidoglycan. More thick than traditional gram positive cell wall made up of a modified peptidoglycan.
Every second murnac is modified to form a delta lactam ring, this cannot be modified with side chain (so doesn’t get amino acids attached to it) only 1 in 4 sugar residues are cross linked rather than 1 every 2 so it’s a more flexible peptidoglycan layer. When a cell comes to germinate - has enzymes (lytic hydrolases) that can specifically recognise the structure which means the spore can break down cortex peptidoglycan.
Then there’s the original cell wall/ primordial cell wall
Spore has two membranes: the original membrane and the second membrane on the outside of the cortex
Core contains a high concentration of CA-DPA and import of that into core of spore drives out water and dehydrates the core, leads to it being enzymatically inert (metabolically) - structures are less suseptible to enzymatic damage as water is required for most reactions
DNA is tightly packaged with soluable proteins - protects from radiation damage and UV damage
What are the stages of sporulation
When triggered a septum is placed (nearer one pole: assyemtric cell division). Larger mother cell and smaller forespore.
The mother cell engulfs the forespore, similar to phagocytosis - membrane is stretched around the forespore. You get a double membrane spore structure. Between these two membranes you get synthesis of the cortex peptidoglycan and extra membrane on the outside derived from the mother cell. On the outside of that membrane, the protein coats are layed down and become chemically cross linked
When spore is completed- mother cell lyses releasing spore into the environment (single spore released into the environment)
What is the gene regulation involved in this process
You get sequential activation of all the genes and its important that we retain this sequential activation. All of the processes have to happen in the right order which is controlled by a sigma factor cascade. Cascade of sigma factors active in forespore and mother cell that activates sequentially because they control a subset of genes, the subset of genes they control get expressed.
By controlling the order of the sigma factors, we control the different activations of the different sigma factors that are expressed and then control the order sequence of events that correctly regulate the spore.
Spo0A- master regulator (response regulator)- when its phosphorylated by kinases, its activated and sporulation starts. Its active at stage 0. The gene designation roughly tells you where this happens in the cascade.
Bacillus sporulation process
Differentiation of the mother cell compartment and the fore spore compartment (sigma f is expressed in the fore spore and pro sigma e (an inactive form of sigma e in the mother cell. Both of these are expressed under control of the Spo0A/ expression - 4 stages of gene expression - well understood.
How is C. difficile different to Bacillus
C. diff is a bit messier: don’t fully understand. A few kinases have been identified that are able to phosphorylate spo0A- don’t know what they respond to. We do know phosphorylated Spo0A leads to expression of sigma F in forespore and expression of pro-sigma e in the mother cell
Sigma f is not involved in the maturation of sigma E. Sigma f is also not required for sigma G in the forespore. We don’t know how the timing is ensured. Process seems to be different to bacillus. We don’t know how this order is maintained in c. diff. Sigma E does seem to be involved in expression of pro sigma K. Cross talk doesn’t happen in c. diff - process is less neat- probably because we don’t understand it.
What does FM464 show in phase contrast microscopy
floursecent membrane stain - makes an important point. The assymetric septum is more brightly labelled than the outside of the cell because there’s two membranes (floursecent intensity is doubled).
What does Dapi show in phase contrast microscopy
Stains DNA- both mother cell and fore spore have DNA - forespore so small, genome has to squeeze into small space (compacted so get more fluorescence)
What do germinants trigger
Upon sensing a germinant 90min to produce a vegetative cell
When germinants trigger, the first thing that happens is the spores rehydrate
Outgrowth occurs: extends out of the spore to form a new vegetative cell
What are the germinants involved in C. diff
Primary germinant of c. diff is taurocholate- mix of cholic acid and the amino acid taurine
Taurocholate is a bile acid.
Primary bile salts are synthesised in the liver
Stored in the gall bladder and released into the duodenum when you eat (they’re detergents)
Bile salts solubalise and emulsify fats – critical for uptake of fats and fat-soluble vitamins
Secondary bile salts produced via bacterial metabolism in the colon- degradation products of the primary bile salts from bacteria in intestines. Bacteria see bile salts as nutrients and break them down for their own uses.
