L9. C. Diff Pathogenesis

Question 1

What does C. diff stand for?

Answer 1

Clostridioides Difficile

Question 2

What type of bacterium is Clostridioides difficile?

Answer 2

Gram positive, spore forming, anaerobic bacterium.

Question 3

How does atmospheric oxygen affect Clostridioides difficile?

Answer 3

Atmospheric oxygen kills it immediately; it cannot survive in O2 at all.

Question 4

When did Clostridioides difficile evolve?

Answer 4

It evolved before the great oxidation event, living in oxygen-deprived niches 2.5 billion years ago (pre-dates oxygen on the planet).

Question 5

What spectrum of diseases does Clostridioides difficile cause?

Answer 5

It causes a spectrum of diseases collectively known as CDAD (C. diff associated diseases caused by CDI/ C. diff infection).

Question 6

What are the characteristics of diarrhoea caused by C. diff infection?

Answer 6

> Self-limiting: Resolved within a few days.

> Recurring: In a small number of cases, it becomes frequent every few weeks, and the infection gets worse with each occurrence.

Question 7

What are the immunological complications of C. diff infection?

Answer 7

> Two major immunological complications are:

1. Pseudomembranous colitis
2. Toxic megacolon

Question 8

What are 3 symptoms and consequences of pseudomembranous colitis if left untreated?

Answer 8

1. Yellow plaques, blisters filled with neutrophils infiltrate the tissue of large intestines causing Plaque build-up on colon surface.
2. Colon wall completely compromised can lead to perforation, causing intestine contents to leak into the abdominal space, leading to sepsis.
3. It is fatal if untreated and is rare but severe.

Question 9

What are the symptoms and consequences of toxic megacolon and how is it treated?

Answer 9

> Rare but severe.

> Intestines can pop out of surgical wounds due to swelling.

> Surgery is the only treatment, which may involve removing the whole colon, requiring a colonoscopy bag.

> Patients often die in surgery.

Question 10

What is the mortality rate of pseudomembranous colitis and toxic megacolon in the UK and globally?

Answer 10

These conditions are rare but responsible for 2000 deaths a year in the UK and probably hundreds of thousands around the world.

Question 11

What is the leading cause of hospital-acquired infection worldwide?

Answer 11

Clostridioides difficile (C. difficile).

Question 12

Which populations are most commonly affected by C. difficile infections?

Answer 12

Most commonly seen in the elderly, but increasingly seen in the community and younger populations (including children and pregnant women).

Question 13

Can C. difficile infect animals? If so, which ones?

Answer 13

Yes, C. difficile can also infect animals such as pigs, cattle, horses, and chickens.

Question 14

What are the associated healthcare costs of C. difficile in the United States and Europe?

Answer 14

> United States: $3.2 billion per year.

> Europe: €3 billion per year.

Question 15

Why do C. difficile infections increase healthcare costs?

Answer 15

Due to extra time spent in the hospital, extending stays by around 10 days, and rooms being out of action for hours to days to stop transmission.

Question 16

What is an issue with current C. diff data?

Answer 16

There is no current data in the UK, and EU data is needed.

Question 17

Describe the disease progression of C. diff in 6 steps

Answer 17

1. Patient receives antibiotic(s)
   >Antibiotic associated diarrhoea
   >95% of patients have antibiotics 2 weeks before symptoms
2. Alterations in normal gut flora
   >Antibiotic induced dysbiosis allows C. diff to colonise the niche and grow.
3. Infection with C. difficile spores
   >Resistant spore form (metabolically inactive), as is an anaerobe needs a spore to survive in environment, spreads to other people and starts growing.
4. Spores germinate in gut forming vegetative cells
5. Cells multiply, produce toxins and sporulate. Spores excreted
   >Toxins: TcdA and TcdB which cause symptoms.
6. Causes more infections with spores from diarrhoea.
   >Spores are almost impossible to kill.

Question 18

How long does the germination process take in C. diff?

Answer 18

90 mins

Question 19

Describe evidence that the microbiota plays a role in C. diff infection

Answer 19

Microbiota bounces back within 2 weeks in mice and as this happens then C. diff is removed; this is probably what happens in humans.

Question 20

When was Clostridioides difficile first isolated and under what name?

Answer 20

1935, isolated from the human infant gut as Bacillus difficile.

Question 21

What is a notable characteristic of C. diff colonization in young babies and animals?

Answer 21

Most young babies are colonized with C. diff asymptomatically, producing toxins without causing diarrhea, a pattern also found in young animals.

Question 22

When was C. difficile established as a major cause of antibiotic-associated diarrhea and pseudomembranous colitis?

Answer 22

1978.

Question 23

What other bacteria can cause diarrhea after antibiotics, though pseudomembranous colitis is mainly caused by C. diff?

Answer 23

P. colitis.

Question 24

How was C. difficile recognized between 1980 and 2000, and what are its major virulence factors?

Answer 24

It was recognized as a gut commensal bacterium in about 5% of healthy adults. Toxins A and B were characterized as major virulence factors.

Question 25

What limitation in screening was noted between 1980 and 2000 for C. difficile?

Answer 25

There was no genetic screening available at that time.

Question 26

What happened in 2003 regarding C. difficile in Quebec and Philadelphia

Answer 26

Epidemics occurred due to new fluoroquinolone-resistant "hypervirulent strains" (NAP1/027).

Question 27

What happened in 2008 regarding C. difficile strains in hospitals, and what was their origin?

Answer 27

Other hypervirulent strains (078) appeared in hospitals, previously associated with animal infections, indicating a zoonotic spillover.

Question 28

What was the major change in the classification of C. difficile in 2016?

Answer 28

It was renamed Clostridioides difficile to reflect the new understanding of genomic variation in the wider Clostridia family, with Clostridia being re-characterized into different genera.

Question 29

What were the trends and significant events in C. difficile-related deaths in England and Wales from 2001 to 2012?

Answer 29

1. Initial undercount due to lack of screening. 2. Deaths decreased to around 2000 by 2012 and remained stable with a slight increase. 3. 2007 saw an outbreak from hypervirulent strains from America, with a decline due to NHS measures (Ribotype 037/ NAP1) 4. Deaths and infections continue to grow significantly in countries with less funded healthcare systems, a major issue in the US.

Question 30

What was the dominant infection strain of C. difficile in the UK in 2001, and how did it change by 2011?

Answer 30

1. 2001: 001 strain dominant. 2. 2004-2005: Outbreak of new 027 (NAP1) strain. 3. By 2007: Over half of UK infections caused by 027 strain, 001 strain down to 8.7%. 4. By 2011: NHS efforts reduced 027 cases to 12.4%.

Question 31

What is the current status of the 027 strains of C. difficile in England, and what has changed?

Answer 31

The 027 strains are under control, but there is now a massive variation of C. diff strains. The strains labeled "others" indicate a wide collection of random C. diff strains, suggesting little evidence of hospital transmission.

Question 32

What does the presence of many different C. diff strains in hospitals indicate?

Answer 32

It indicates that the infections are coming from a separate reservoir, not from hospital transmission, as hospital transmission would show lots of patients with the same strains.

Question 33

What does a decrease in same-strain infections in hospitals suggest?

Answer 33

It suggests that hospital cleaning efforts are working.

Question 34

Despite hospital cleaning efforts, what remains a problem with C. diff infections?

Answer 34

There is a baseline number of infections occurring from outside of the hospital.

Question 35

What is the strain responsible for the North American outbreaks, and what is its name in Europe?

Answer 35

The strain is NAP1 (North American Pulse-field electrophoresis Type 1), known in Europe as Ribotype 027.

Question 36

What is a notable characteristic of the Ribotype 027 strain

Answer 36

It is fluoroquinolone-resistant.

Question 37

What are 4 key characteristics of the "hyper-virulent" 027/NAP1 (ribotype 027) strains of C. difficile?

Answer 37

1. Previously rare. 2. Now fluoroquinolone-resistant (suggests recent acquisition). 3. Associated with large outbreaks (more transmissible). 4. Associated with more severe disease (hyper-virulent), becoming more harmful.

Question 38

What are the 4 specific characteristics of the 027/NAP1 (ribotype 027) strains?

Answer 38

1. Produces more spores, making it more transmissible. 2. Produces more toxin in vitro, leading to more severe disease. 3. Produces a third toxin – binary toxin (CDT). 4. Contains an 18 bp deletion in tcdC, which upregulates toxin production.

Question 39

What bias is noted in the collection of C. difficile strains?

Answer 39

Places not collected have bigger threats than C. diff, so they aren't being worried about as much, but 027 strains are found everywhere and have gone global.

Question 40

What is trehalose sugar, and why is it significant in relation to C. difficile?

Answer 40

Trehalose sugar is mainly a manufactured sugar used to make ice cream smoother and has become cheaper in the past 20 years due to enzymatic production from plant starch. C. diff, particularly the 027 lineage, is very good at metabolizing trehalose.

Question 41

What mutation in epidemic ribotype 027 increases its virulence?

Answer 41

A point mutation in treR, a regulator of sensing trehalose, switches the enzymatic pathway for degrading it and using it as a carbon source. This mutation makes the strain 500 times more sensitive to trehalose and using it as a carbon source.

Question 42

What happens when the treA gene (trehalose degradation enzyme) is deleted in ribotype 027 in mice?

Answer 42

The virulence is massively attenuated, but supplementation with trehalose enhances virulence, showing that trehalose in food causes increased virulence.

Question 43

When do most C. difficile infections occur?

Answer 43

Most C. difficile infections follow treatment with antibiotics.

Question 44

Why does an intact microbiota prevent colonization/infection with C. difficile?

Answer 44

>Microbiota are natural occupiers and don't want to give up their niche, so they compete with most pathogens infecting the colon as microbiota are evolved to live there. 1. They effect bile acid metabolisation, and therefore mediate sporulation and germination of C. diff (bile salts = germinant for C. diff) 2. They outcompete pathogens by consuming available food. 3. They produce metabolites which C. diff doesn't like, such as SCF. 4. Bacteria directly produce bacteriocins which kill C. diff (antibacterial). 5. Microbiota modulate the host innate immune response, producing antimicrobial peptides to which C. diff is sensitive.

Question 45

What is a prerequisite for C. diff infection (CDI)?

Answer 45

Dysbiosis (an imbalance in the gut microbiota) is a prerequisite for CDI (C. Diff Infection)

Question 46

How does the microbiota recover after a C. diff infection is cleared?

Answer 46

The microbiota recovers naturally as the infection is cleared.

Question 47

How can the recovery of microbiota be exploited to treat C. diff infection and what is evidence for this?

Answer 47

Faecal microbiota transplantation from a healthy donor is 90% effective for treating C. diff infection.

Question 48

Which animal model was previously used for C. difficile infection but is now barely used?

Answer 48

Golden Syrian Hamsters.

Question 49

Which animal model is mostly used now for C. difficile infection studies?

Answer 49

C57BL/6 Mice (standard lab mouse).

Question 50

Describe the hamster model for studying C. difficile infection, what is was used to study specifically and why it was stopped being used.

Answer 50

>Clindamycin-treated hamsters are challenged with C. difficile spores. >C. difficile infection is lethal within a few days, unlike in humans. >Used to study the acute phase of C. difficile infection where toxins are key virulence determinants. >Model went out of use due to ethical concerns (animals died), inaccuracy (humans don’t die in 3 days after infection), and hamsters not being commonly used species in biology.

Question 51

What are the advantages of using the mouse model (C57BL/6 Mice) for C. difficile infection studies?

Answer 51

1. Cheaper as mice can be caged together (social, unlike aggressive hamsters). 2. Availability of reagents and knock-out animals for mice. 3. Less lethal for mice (C. diff infection).

Question 51

What are 3 reasons to why the hamster model no longer commonly used for C. difficile infection studies?

Answer 51

1. Ethical concerns as animals died. 2. Doesn't replicate what happens in humans accurately (humans don’t die in 3 days after infection). 3. Hamsters are not commonly used species in biology, unlike mice which have many monoclonal antibodies and other important reagents for use in the lab

Question 52

What are the advantages of using the mouse model (C57BL/6 Mice) for C. difficile infection studies?

Answer 52

1. Cheaper as mice can be caged together (social, unlike aggressive hamsters). 2. Availability of reagents and knock-out animals for mice. 3.Less lethal for mice (C. diff infection)

Question 53

Describe how the mouse model is used for studying C. difficile colonization and what it is specifically used to study.

Answer 53

>Clindamycin-treated mice are challenged (become susceptible to infection) with C. difficile spores. >By giving a cocktail of antibiotics instead, it makes C. diff infection more severe and can be tailored to mimic different human infections. >High number of spores are shed in the feces, referred to as a super-shedder state. >After cessation (microbiota recovers) of antibiotic treatment, the number of C. difficile spores in the feces decreases, but a subsequent dose of clindamycin returns animals to the super-shedder state. >This model is a good replication of relapsing C. diff in humans.