WEEK 11: CLOSTRIDIAL INFECTIONS Flashcards
Are vaccines often toxin based for C.perfringes?
- YES
What are some characteristics of genus Clostridium?
- Gram +ve rods
- Obligate anaerobes
- Form heat resistant endospores (heated=activated, - endo= within bacterial cell)
- Common inhabitants of the GI tract
- Important in agriculturs (soil maintenance–> break down organic matter) and industry (biofuels)
- Important human and animal pathogens (only under certain circumstances)
What are two ways to culture Clostridium if it is an anaerobe?
- Anaerobic jars
- Anaerobic chamber
When does clostridium form endospores?
- Under adverse environmental conditions (e.g. presence of O2) act as survival mechanism
What are the clostridium spores characterized based on?
- The position
- The size
- The shape
What type of spores do most clostridium have?
- OVOID subterminal spores (OST)
What kind of spores do C.tetani have?
- Round terminal spores (RT)
What does the exosporium that clostridium have allow them to be?
- Allow them to be sticky. So spores of epidemic strains are stickier.
What do the pathogenic Clostridia produce?
- Potent protein toxins
- E.g. C.perfringes–>gas gangrene
- C.tetani–> tetanus
- C.botulinum–> botulism
What are the neurotoxic Clostridia?
- C.tetani and C.botulinum (tetanus and botulism)
What are the enterotoxic Clostridia?
- C.perfringes
- C.difficile
What does Clostridium Tetani cause and what does this produce?
-Causes tetanus and produces the tetanus toxin (neurotoxin)
What is the pathogenesis of tetanus?
Entry of spore into DEEP wound (these cut off blood supply to tissue so it can be anaerobic)
Germination of spore in anaerobic environment
Production of toxin by growing bacteria which is released when they die
Toxin binds to nerve endings and it is TRANSLOCATED into nerve cells
This INHIBITS NT release –> blockage of the muscle relaxation pathway
What effects does tetanus lead to?
-Uncontrolled stimulation of muscles —>tension, cramping twisting of muscles, spasms and convulsions, rigid paralysis, death from spasms of the diaphragm and respiratory muscles
What is an effective preventive measure for tetanus?
Vaccination using the tetanus toxin
What does Clostridium botulinum cause?
- Causative agent of botulism
- Food-borne disease
- Commonly from contaminated canned foods that have been inadequately heated
What is the pathogenesis of Clostridium botulinum?
Spores germinate in food and toxin is produced during vegetative growth
Pre-formed toxin (BoNT) is INJESTED with food
Toxin localises in neuromuscular junction–> BLOCKS the release of NT
-C.botulinum produces a lot of gas when it grows (gas gangrene)
What does Clostridium botulinum result in?
- Uncontrolled relaxation of muscles
- Symptoms within 18-24 hours of ingesting toxin
- Blurred vision, difficulty speaking, muscle weakness, nausea, vomiting
- Flaccid paralysis
- death due to cardiac or respiratory failure
Is there a vaccine available for Clostridium botulinum?
NO
- But can treat it with antitoxin if administered quickly
What are the therapeutic uses of botulinum toxin?
- BoNT used to treat severe focal dystonias muscle spasms and facial tics
- BoNT used in cosmetic industry removal of wrinkles
- Very safe and effects last 1-4 months
Where is BoNT injected into for theraeutic use?
- Striated muscle –> leads to REVERSIBLE denervartion of NMJ
Can you get Igs against the toxin which reduce the effectiveness of it?
- YES
What is the location of C.perfringes?
- IN the gut of everyone
Can C.perfringes cause food poisoning?
- YES –> Present in meat that has been heated and cooled slowly
- Ingested in HUGE numbers
Does C.perfringes prodce an enterotoxin?
- YES
- Bacteiral cells sporulate in intestine
- Production of enterotoxin is associated with sporulation
How long does it take for the onset of symptoms in C.perfringes?
- 8-16 hours –> then watery diarrhoea, nausea, abdom cramps
Is treatment usually required for C.perfringes?
- NO but antibiotics can be prescribed
What does C.pefringes cause in humans?
- Food poisoning and gas gangrene
What does C.perfringes cause in animals?
- Enterotoxanaemic diseases
What toxin do ALL the strains of C.perfringes produce?
- Alpha toxin–> difficult to make vaccines
What are the predisposing factors that cause C.perfringes infections in animals (or make them susceptible) ?
- Antibiotics
- Feeding changes (changes in microbiome)
- Change of weather conditions (growing in field) –> will kill animals in 12-18 hours
Which toxins does the C.perfringes type C infection produce and which is the most important?
- Produces the alpha and beta toxin BUT the beta toxin is the most important in C.perfringes infection
What is the Beta toxin sensitive to?
- Trypsin
What does the beta toxin cause in humans?
-Enteritis necroticans (pig-bel) role of the trypsin inhibitor in sweet potatoes (inhibits the breakdown of the Beta toxin)
What is the pathogenesis of infection for C.perfringes type C?
beta toxin produced in the gut; gut trypsin degrades beta toxin. In type C infections:
- Low trypsinL Beta toxin remains active
- Intestinal necrosis
- Beta toxin and other toxins absorbed
- Toxins act on distant organs
What are the predisposing factors for the type D infection (c.perfringes)?
- Carbohydrates in diet (sudden changes)
- Bacterial contamination levels (farm)
- Immune status (against the ϵ toxin )
Which toxins does type D produce and what is the main one? (C.perfringes)
- Alpha, and Epsilon
- Epsilon (ϵ) is the MAIN ONE
What is the pathogenesis of the type D C.perfringes infrection?
- Pro-ϵ-toxin: prodcued in intestine
- Activated by trypsin/other proteases
- Increases intestinal permeability.
- Absorbed systemic circulation.
- Endothelial cells of brain, kidney etc.
- Effects of brain lead to neurological signs –> brain lesions in cattle
What are the KEY concepts to remember about C.perfringes? (5 things)
- Strain colonises
- Produces enteric toxin
- Enteric toxin PERMEABILISES the gut
- Toxins and other factors are absorbed via the ‘leaky’ gut
- Toxins act on DISTANT organs
What is gas gangrene (clostridials myonecrosis)?
- injured tissue becomes contaminated with spores
- if tissue is anaerobic spores germinate and bacteria rapidly grow
- extensive bacterial growth is observed
What does C.perfringes type A cause?
- Gas gangrene
What are the mechanisms of the α toxin?
- Phospholipase–> disrupts host-cell plasma membranes
- Extensive destruction of cells and tissues
What are the symptoms of gas gangrene?
- Severe pain
- Edema
- Muscle necrosis
- Lesions and blackening of skin–> TREATMENT= amputation usually
Does each part of the GI tract have a different microbiota?
- YES
Where does C.difficile arise/live and what is this environment like?
- In the Descending colon
- Environment is anaerobic
What 3 things does the microbiota change with?
- Age
- Diet
- Health
Is the microbiota acquired after birth?
- YES
How does the destruction of normal microbiota by antibiotics affect their non specific immunity?
- Leaves the host vulnerable to infections by opportunistic bacteria
Does C.difficile occur by antibiotics used for an unrelated condition?
- YES
During a C.difficile infection, what happens to the colonic structure, the integrity mechanisms, and the stem cells and niche?
- Colonic structure is disrupted
- Integrity mechanisms are purtubed (disrupted) –> Adherens-junctions (cell-to-cell contacts), Erzin (cell polarity)
- Stem cells and niche are DAMAGED –> repair capactiy disrupted
- Combination of these result in severe damage and disease
Id C.difficile an urgent humans threat?
- YES
- Hypervirulent strains emerged around 2005 and caused epidemics
What drove the Hypervirulent C.difficile strains in 2005 ?
- Over use of Fluoroquinolone antibiotic –> resistant strains of C.difficile
What enhances the dissemination of C.difficile strains?
- Travel
DO C.difficille infections just come from healthcare settings?
- NOOO
For C.difficile (as with many other bacteria) is there likely a close relationship b/w animal and human strains and transmission?
- YES –> One health problem
What 5 things does the microbiota derive?
- Supply of nutrients
- Stable environment
- Constant temperature
- Protection
- Transport
What does the host derive?
- Some nutritional benefit
2. Prevention of colonisation of pathogens
Is C.difficile acting as a parasite?
-YES because microorganism that benefits at the expense of the host
Is C.diffiile NATURALLY present in the gut?
-YES
What are 4 ways the normal microbiota inhibits the colonisation of pathogens?
- Producing metabolic products –> FAs, bacteriocins (bacterial antibiotics)–> inhibit the growth of many pathogens
- Adhering to target host cells–> Prevent pathogens from colonising
- Depleting nutrients essential for the growth of pathogens
- Stimulating the immune system –> e.g. no microbiotia= no mucous production
What is the term for destruction of the normal microbiota through the use of antibiotics?
-Dysbiosis
What happens to C.difficile to cause the production of the toxin?
- It overgrows in the intestinal tract
- Antibiotic associated colitis (so if you take antibiotics, this leaves opportunity for C.difficile to flourish! This then leads to the colitis)
What are the characterisitics of C.difficle?
- Leading cause of infectious diarrhoea in hospitals worldwide
- Also a problem in the community (pigs, cattle, horses)
- STRICT anaerobe and spore former –> spore are - CRITICAL for infection (persistence is problem)
- C.difficile ONLY colonises gut if normal microbiota is DISRUPTED (via antibiotics)
In C.difficile, are the spores critical for infection?
- YES
Is C.difficile a strcit anaerobe and spore former?
- YES
Does C.difficle only colonise the gut if the normal microbiota disrupted?
- YES
Is a patient resistant to C.difficile if the normal microbiota is not disrupted by antibiotics?
- YES
What is the main pathway/steps in the formation of C.difficle with antibiotic use?
- When antibiotic treatment commences, infection with resistant strain is more likely while the antibiotic is being administered
- When antibiotic treatment ceases, microbiota remains disturbed during which patients CAN be infected with resistant OR suscpetible C.difficile
- After microbiota recovers, colonisation resistance to C.difficile is restored
What is the C.difficle infectious cycle?
- Reduction of normal gut microflora
- Ingestion of spores
- Spores survive in stomach
- Bile salts in the small intestine trigger germination events
- Anaerobic environment of colon supports vegetative growth and toxin production
- Spore shedding
Which symptoms does C.difficile-mediated pseudomembranous colitis produce?
- yellowish plaques of fibrin, mucous and inflammatory cells overlay the normal intestinal mucosa
What does the C.difficile disease pathology result from?
- toxin production
How is C.difficile diagnosed?
- PCR or ELISA
What are the major virulence factors in C.difficile?
- Toxins A and B –> TcdA and TcdB
- But mainly TcdB
Are the C.difficile toxins located on a pathogenicity island?
- YES –> found in all toxogenic strains
What are the specific actions of toxins A and B?
- Catalyse transfer of a glucose moiety onto the Rho family of GTPases (Rho, Rac, and Cdc42) INSIDE target cells
- Protein inactivation–> Cell death from the breakdown of actin filament network, which is integral for the maintenance of cell cytoskeletal structure
What does the integrity of the intestinal epithelium rely on?
- Cellular polarity
- Formation and maintenance of tight junctions
- Renewal of the stem cell population and maintenance of the stem cell niche
Is cellular polarity important for intestinal homeostasis?
-YES
What is both important for apical integrity AND can be used as a marker for cellular polarity?
-Erzin –> immunofluorescent staining and confocal microscopy
What is the most rapidly proliferating tissue in adult mammals and is it constantly replaced in adult mammals?
- The gut
- YES constantly replaced
How is the intestinal integrity maintained?
-Stem cells at base of epithelium give rise to new cells and MIGRATE to crypt surface and sloughed off through apoptotic death
How is the maintenance of the intestinal eputhelium process affected during C.difficile infection?
It DAMAGES colonic stem cells
What was used to determine that C.difficile infection affects stem cell function AND gut repair capacity?
-Organoid culturing –> 3D organ-bud grown in vitro from stem cells that forms a ‘mini gut’–>ex vivo
What does a diagram comparing the organoids from no C.difficle infection and the organoids from a C.difficle infection tell us?
-That infection affected PLOARITY, TIGHT JUNCTIONS, and deep down in crits (damages stem cells–> affects repair capacity of gut)
Is there on single reason why C.difficile is debilitating and why people take a long time to get over it?
- NO
- Combined reasons
From looking at healthy tissue and unhealthy tissue then producing colonic organoids, which toxin was the devastating effect primarily found to be from?
-TcdB (Toxin C difficile B)
What symptom does C.difficile have that other gut infections such as IBS and IBD have?
‘leaky gut’ –> damaged junctions b/w cells –>this leads to spores and other gut contents disseminating beyond gut in severe disease
What are the current treatments for C.difficle?
- Discontinuation of antibiotic, and fluid replacement
- More antibiotics –> Oral metronidazole and/or vancomycin BUT relapses occur (bc. microbiota depleted)
- Other antibiotics are undergoing trials or have been approved (e.g. fidaxomicin–> narrow host range) BUT $2500 for course
What are treatments for C.difficile that are currently under consideration?
- NON-ANTIBIOTIC treatments –>
o Probiotics
o IV IgG antibodies (containing human anti-toxin IgG)
o mAB for passive immunotherapy
o Passive polyclonal immunotherapy (cow colostrum; egg yolk Igs)
o Faecal transplant therapy
What is the analogy of fecal transplant therapy in terms of grass?
- Rolling a turf of grass over the old and damaged one
What is an issue with Fecals transplants?
- some people can have drug resistant bacteria–> someone as a result died from a transplant
How does the ‘One Health’ relate to C.difficile?
- It is UNDER RECOGNISED cause of disease in animals –> neonatal pigs, beef and dairy calves, horses and companion animals
- No estimates on economic burden
Can C.difficile be detected in meat and meat products and if so, what does this suggest?
- Yes
- Suggests foodborne transmission to humans
- Also can be detected in water runoff and compost –> suggests other transmission portals
