Clostridium Flashcards
Clostridium
gram positive rods
obligate anaerobes
can form heat resistant endospores
are common inhabitants of gastrointestinal tract
play an important role in biofuels and agriculture
Are important human and animal pathogen
Anaerobic culture techniques
use a gas chamber containing a mixture of carbon hydrogen and nitrogen
Ensure the walls of chamber have very low oxygen permeability so oxygen is unable to get in
Ensure all equipment is placed inside chamber such as culture media and carry out manipulations inside chamber
Use entry port for oxygen evacuation + entry and exit of materials
Spores
Clostridial Endospores
• Clostridium form endospores under adverse
environmental conditions, such as the presence of
oxygen
• Spores are a survival mechanism
• Spores are characterised on the basis of position,
size and shape, can be used for identification
How is spore produced
There are two cycles - vegetative cycle and sporulation
Vegetative cell are when bacterial cells are dividing and doubling population
Sporulation is triggered in adverse environmental conditions
1. Polar division to form prespore, septum and mother cell compartment.
2. All DNA from cell is pumped into the prespore.
3. Layers form around spore such as cortex and cell wall and cell membrane.
4. There is the formation of a spore coat around the spore which acts as armour
5. cell lyses, releasing spore into the environment
6. spore will germinate when conditions are right again and reenter vegetative cycle
Bacterial endospore structure
intermembrane, cortex, outermebrane, spore coat, exosporium (not always there)
Types of Clostridium pathogens
The Pathogenic Clostridia Neurotoxic C. tetani C. botulinum Enterotoxic C. perfringens C. difficile Histotoxic C. perfringens
Common feature: produce potent protein toxins
Clostridium tetani
Neurotoxic Clostridia
• Clostridium tetani
— causative agent of tetanus
— produces tetanus toxin
• neurotoxin
— pathogenesis involves
• entry of spore into deep wound as in deep wounds blood circulation is cut off and there is no oxygen - anaerobic environment
• germination Of spore in anaerobic environment
• production of toxin by growing bacteria which is
released when they die
• toxin binds to nerve endings and is translocated into
nerve cells
• inhibits neurotransmitter release
• blockage of muscle relaxation pathway
Tetanus toxin effects
Pathogenesis of Tetanus • Results in uncontrolled stimulation of muscles —tension, cramping twisting of muscles —spasms and convulsions —rigid paralysis —death from spasms of the diaphragm and respiratory muscles • Vaccination using tetanus toxoid is an effective preventative measure
Clostridium botilunum
Neurotoxic Clostridia
• Clostridium botulinum
— causative agent of botulism
• food-borne disease
• commonly acquired from contaminated canned foods
that have been inadequately heated
— pathogenesis
• spores germinate in food and toxin is produced
during vegetative growth
• pre-formed toxin (BoNT) is ingested with food
• toxin localises in neuromuscular junction
• blocks release of neurotransmitter
Effects of botulin toxin
Pathogenesis of Botulism • Results in an uncontrolled relaxation of muscles — symptoms within 18 to 24 hrs of ingesting toxin — blurred vision, difficulty swallowing and speaking, muscle weakness, nausea, vomiting — flaccid paralysis — death due to cardiac or respiratory failure • No vaccine available — can treat with antitoxin if administered quickly
Clostridium perfringens
Enterotoxic Clostridia — human disease
• Clostridium perfringens
— aerotolerant anaerobe; forms spores
— can cause food poisoning
• present in meat that has been heated and cooled slowly
• ingested in huge numbers
— produces an enterotoxin
• bacterial cells sporulate in the intestine
• production of enterotoxin is associated with sporulation
— onset of symptoms after 8 to 16 hours
• watery diarrhoea, nausea, abdominal cramps
C: perfringen disease in animals
C. perfringens toxins and disease
• Enteric pathogen of humans and animals
— humans: food poisoning, gas gangrene
— animals: enterotoxaemic diseases
• Virulence largely attributed to the productionof up to >22
toxins, particularly in animal diseases
• However, not all isolates express all toxins
• Vaccines are often toxin-based
What do you need to get Clostridium infection?
Predisposing factors are critical for susceptibility
Type D C.pefringens
C. perfringens type D infections
Type D strains colonise the gut. Predisposing factors:
I - Carbohydrates in diet — sudden changes
2 - Bacterial contamination levels
3 - Immune status - vaccination
Effects of C.perfringens toxin
Pathogenesis of Type D infections Pro-E-toxin: produced in intestine activated by trypsin/other proteases increases intestinal permeability absorbed systemic circulation endothelial cells of brain, kidney etc.
Gas gangrene
Histotoxic Clostridia — human disease
• C. petfringens
— causes gas gangrene (clostridial myonecrosis)
• injured tissue becomes contaminated with spores
• if tissue is anaerobic spores germinate and bacteria
rapidly grow
• extensive bacterial growth is observed
Alpha toxin
Histotoxic Clostridia
• C. perfringens
— produces a-toxin
• phospholipase
• disrupts host-cell plasma membranes
• extensive destruction of cells and tissues
— symptoms include severe pain, edema, muscle
necrosis
• Characteristic lesions and blackening of the skin
associated with gas gangrene are seen
• Treatment is radical and usually involves amputation
of the affected tissue/limb
Normal microbiota
Normal host microbiota
Internal tissues usually free of microorganisms.
Surface tissues (skin, mucous membranes) in
contact with environment, therefore
colonised by bacteria, yeasts, fungi.
Mutualistic relationship - both host and
microbiota derive benefit.
How do microbiota and host benefit each other
Normal host microbiota
Microbiota derives:
supply of nutrients stable environment constant temperature protection transport from one host to another Host derives: I. 2. some nutritional benefit prevention of colonisation of pathogens
Non specific immunity caused by microbiota
keep potentially harmful opportunistic pathogens in
check and inhibit the colonisation of pathogens by:
1. Producing metabolic products: Fatty acids,
bacteriocins, etc. inhibit the growth of many pathogens.
2. Adhering to target host cells: Prevent pathogens
from colonising.
3. Depleting nutrients essential for the growth of
pathogens.
4. Stimulating the immune system so it is primed to
fight pathogens.
Impact of antibiotics on microbiota
Non-specific immunity by normal microbiota
Destruction of normal microbiota by use of
antibiotics leaves host vulnerable to
infections by opportunistic microbiota.
Examples: Candida and Clostridium difficile.
- Held in check by normal microbiota.
- Not killed by antibiotics (why?)
* Candida —Y thrush.
* C. difficile overgrows intestinal tract —Y toxin
antibiotic-associated colitis.
About C Difficile
Enterotoxic Clostridia Clostridium difficile: an opportunistic pathogen • Leading cause of infectious diarrhoea in hospitals worldwide. • Also a problem in the community and animals (pigs, cattle, horses) • Strict anaerobe and spore former — spore are critical for infection; their persistence is a problem. • C. difficile only colonises gut if normal microbiota is disrupted — usually via antibiotics. Note Clostridium difficile renamed Clostridioides difficile recently
Antibiotics and C difficile
Antibiotics and C. difficile infection I. patient resistant to CDI if normal microbiota not disrupted by antibiotics 2. When antibiotic treatrrmt commences. infection With resistant strain more likely while antibiotic is being administered 3. When antibiotic treatment ceases, microbiota remains disturbed during Which patients can be infected With resistant or susceptible C. dimcile 4. After microbiota recovers, colonisation resistance to C. drmcile is restored. C. difficile is a problem of antibiotic use.
C difficile infectious cycle
spores are ingested
spores survive acidity of stomach and go down to the intestine
bile salts trigger germination of spores
anaerobic environment of colon supports vegetative growth and toxin production
spores shed in faeces
C.difficile pathology
toxins cause yellowish plaques of fibrin mucus and inflammatory cells to overlay intestinal mucosa
C. difficile toxins
Major virulence factors of C. difficile
Toxins A and B
• Located on a 19.6 kb pathogenicity locus (Pal_oc)
- found in all toxigenic strains, absent in avirulent strains.
• Members of large clostridial glucosylating toxins family
— monoglucosyltransferases that glucosylate Rho GTPases.
• Toxin action results in severe damage to intestinal epithelium.
Intestinal integrity and maintenance
Intestinal integrity and maintenance • The integrity of the intestinal epithelium relies on — Cellular polarity — Formation and maintenance of tight junctions — Renewal of the stem cell population and maintenance of the stem cell niche
Cellular polarity and intestinal integrity
Cell polarity and intestinal integrity
• Intestinal epithelial cells are prototypical
polarised cells
— Distinct lumen-facing apices with microvilli
— Bases anchored to basal lamina
• Cellular polarity is important for intestinal
horneostasis
• Ezrin is important for apical integrity in
intestinal epithelial cells
— Can be used as a marker for cellular polarity
Examined using immunofluorescent staining and confocal microscopy
What does C.difficile do to cell polarity?
Damage cell-polarity colonic surface
Tight junctions and intestinal integrity
• Adherens junctions (AJ)
maintain intestinal integrity
• Connect actin cytoskeleton
between cells- connect the A and B catenin to and A and B catenin of another cell via E cadherin
• Disruption Of junction can
exacerbate colonic
inflammation and cause ‘leaky’ gut - toxins and other factors disseminate beyond the gut into the bloodstream
What does C.difficile do to tight junctions?
It disrupts tight junctions = no B catenin and E cadherin co localization
Stem cell population and intestinal integrity
Intestinal integrity and maintenance • The gut is the most rapidly proliferating tissue in adult mammals and the intestinal epithelium is constantly replaced — Stem cells at the base of the epithelium give rise to new cells — Migrate to crypt surface and are sloughed off through apoptotic death
Does C.difficile damage stem cells?
C. difficile infection damages colonic stem cells
• Organoid culturing was used to determine that C difficile
infection affects stem cell function and gut repair capacity
Organoids are 3-dimensional organ-bud grown in vitro from stem cells, that forms a ‘mini gut’
CDI alters stem cell function and organoid formation - killed stem cells so organoids decreased, and disrupted stem cell function so organoids didn’t grow normally
Thus intestinal repair would require stem cells from adjacent tissue - much longer process and prevents integrity from being maintained: Toxin B has most effect on stem cells
Treating C-difficile
Current C. difficile treatments
• Discontinuation of antibiotic and fluid replacement for diarrhoae
• More antibiotics for infection!
— Oral metronidazole and/or vancomycin - vancomycin is better
• Relapses occur because NM keeps getting disrupted
• Other antibiotics are also undergoing trials or have
been approved, (eg fidaxomicin; narrow host range - doesnt kill NM much so relapse is minimized)
Non antibiotic C difficile treatments
• Probiotics for stimulating growth of microbiota and replenishing microbiota
• Intravenous lgG antibodies harvested from humans who have recovered from infection - IgG antibodies are specific for toxin and can bind to toxin and neutralize it
• Make monoclonal antibodies against toxins for passive immunotherapy
• Make polyclonal antibodies - cow fed with inactivated toxin, antibodies against toxin is harvested from colostrum as milk contains really high concentration of antibodies
- Faecal transplant to replace entire microbiota - entire microbiota is transplanted from one individual to another. However need to screen faeces to ensure no harmful microbes in faeces.
All of these are used after antibiotics usage is discontinued
Where do C.difficile infections come from?
Hospital is a source of transmission, but C difficile infections can also come from consumption of contaminated meat - C difficile can infect animals
C.difficile animal infections
C. difficile infections of animals
• Under-recognised cause of disease in animals
• No precise estimates of economic burden.
• Can be detected in meat and meat products
— suggests foodborne transmission to humans.
• Can also be detected in water runoff and compost
— suggests other transmission portals.
As with many bacteria, it is likely that there is a
close relationship between animal and human
strains and transmission: a “one health” problem.
