Infectious diarrhoea I

Question 1

Broadly describe infectious diarrhoea, risk factors and how it occurs

Answer 1

• Many enteric bacterial pathogens can cause infection followed by toxin-mediated disease.
• Ingestion of contaminated food or water is a common mode of infection of the GIT.
• Diarrhoea caused by enteric pathogens is a major cause of morbidity and mortality worldwide:
  
  • An estimated 2-4 billion episodes of infectious diarrhoea occur each year and are especially prevalent in infants.
  • Overall, rates of infectious diarrhoea are much higher in developing settings due to poor water sanitation, poor access to clean and safe drinking water and undeveloped hygiene practices.
  • Malnutrition, weakened immunity and overall lower health status are key factors contributing to fatal diarrhoeal episodes.
• Underlying mechanisms of infectious diarrhoea differ depending on the pathogen, but generally involve direct or indirect alterations in:
  
  • Ion transport
  • Tight junctions

Question 2

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Describe general mechanisms of diarrhoea

Answer 2

• Normally the GIT has tremendous capacity to absorb fluid and electrolytes:
  
  • 8-9 L of fluid is presented daily to the intestine
  • Only 100-200 mL of fluid is egested via stool
• Enteric pathogens can alter this balance towards net fluid loss, leading to diarrhoeal disease
• Diarrhoea is simply an altered movement of ions and water that follows an osmotic gradient
  
  • i.e. Diarrhoea caused by either an increase in secretion and/or decrease in absorption of fluids and electrolytes; this occurs via either cell membrane transporters or the lateral intercellular spaces (regulated by tight junctions)

Question 3

Describe the three types of diarrhoea and provide examples of causative organisms

Answer 3

Secretory diarrhoea
- Through increased secretion or decreased absorption of fluids and electrolytes
- e.g. Vibrio cholerae, ETEC - disease is mediated by exotoxins in the gut

Osmotic diarrhoea
- Through loss of absorptive surface causing increased osmolarity of intestinal contents and malabsorption
- e.g. EHEC - mediated by attaching and effacing (A/E) lesions on intestinal epithelium

Inflammatory diarrhoea
- Through inflammation causing damage to host tissues resulting in fluid exudation
- e.g. C. difficile - mediated by direct damage to the IEC barrier and disruption of tight junctions

Question 4

Describe bacterial toxins, how they cause damage and types of toxins

Answer 4

• Toxins: Microbial products that injure other cells and/or organisms. Toxins can also induce host immune responses that cause inflammation.
• Bacterial Endotoxin: LPS (lipopolysaccharide) present in Gram-negative bacteria. An intrinsic feature of the bacterium
• Bacterial Exotoxin: Soluble proteins secreted into extracellular environment by some species of Gram-positive and Gram-negative bacteria.
  
  • Toxins can attack different cell types:
    
    • Enterotoxins
    • Neurotoxins
    • Leukotoxins
    • Cytotoxins
  • Can act through specific mechanisms:
    
    • Botulinum toxin (Clostridium botulinum): blocks neurotransmitter release leading to paralysis (botulism)
    • Streptolysin O (Streptococcus pyogenes): haemolysis
    • Pertussis toxin (Bordetella pertussis): alters cell function leading to cell death

Question 5

Compare and contrast microbial intoxication vs infection

Answer 5

Intoxication
- Results when a host ingests pre-formed exotoxin
- Ingested enterotoxins directly cause disease
- Presence of a replicating pathogen is not required for disease
- often requires shorter incubation time eg hours, as pre-made toxin is cause of symptoms

Infection
- Results when a pathogen colonises a part of the body and subsequently causes disease
- Often pathogens will then synthesise toxin which causes toxin-mediated disease
- often slightly longer incubation time eg 1-2 days compared with intoxication (hours)

Question 6

Provide examples of organisms causing intoxication and common food sources

Answer 6

Clostridium botulinum
- Gram-positive bacillus, spore-forming, obligate anaerobe
- Intoxication caused by eating foods containing botulinum toxin

Staphylococcus aureus
- Gram-positive coccus, facultative aerobe
- Intoxication caused by eating foods containing Staphylococcus enterotoxins

Bacillus cereus
- Gram-positive bacillus, spore-forming, facultative anaerobe
- Intoxication caused by eating foods containing haemolysin BL (HBL), non-haemolytic enterotoxin (NHE) and cytotoxin K (CytK)

Foods commonly associated with intoxication:
- Meats (seafood, poultry and others)
- Rice
- Pasta
- Milk
- Cheese

Question 7

Describe V. cholarae, and the clinical features and treatment of cholera

Answer 7

Vibrio cholerae
- Gram-negative, facultative anaerobe, highly motile bacillus with a single polar flagellum (locomotion organelle).
- Persists in natural aquatic reservoirs and infects human host via consumption of contaminated water/food.
- People with access to adequately treated water typically not exposed.
- Areas without adequate water treatment still suffer from epidemic infection.
- Causative agent of cholera, an acute diarrhoeal disease which can be fatal without treatment.
- A type of secretory diarrhoea
- Estimated 3–5 million cases and 100,000–120,000 deaths due to cholera every year.

Cholera: Clinical Features and Treatment
Clinical features
- Acute profuse watery diarrhoea with severe dehydration (can lead to hypovolaemic shock and death if untreated)

Treatment
- Cholera is a readily treatable disease
- Up to 80% of infected people can be treated successfully with prompt administration of oral rehydration solution
- Severely dehydrated patients require administration of intravenous fluids
- May also require adjunctive antibiotics to attenuate duration of diarrhoea, reduce volume of rehydration fluids needed, and shorten duration of V. cholerae excretion

Question 8

Describe the colonisation factors of V. cholerae

Answer 8

• Colonisation is dependent on production of a type IV pilus, toxin coregulated pilus (TCP), which is essential for adhesion to the small intestine.
• N-acetylglucosamine-binding protein A (GbpA) is also required (mediates binding to intestinal mucin).
• Colonisation of the gut is a pre-requisite for the subsequent production of toxins which are directly responsible for the profuse diarrhoea.
  
  • V. cholerae causes toxin-mediated disease

Question 9

Describe V cholerae toxins, structure and function

Answer 9

V. cholerae Toxins
- V. cholerae has 7 different toxins that together result in a particularly severe diarrhoea.
- Cholera toxin (CT), accessory cholera toxin (ACE), NAG-stable toxin, and V. cholerae cytolysin (VCC): affect ion secretion into the intestinal lumen.
- Haemagglutinin/protease (HA/P), repeats in toxin (RXT), and zonula occludens toxin (Zot): promote the loss of barrier function.

Cholera Toxin Structure
- Consists of an A subunit bound to a pentameric ring of B subunits, where the B subunits are responsible for delivery of the A subunit into the host cell.

Cholera Toxin Function
- CT (A subunit): causes Cl− secretion and Na+ absorption, leading to an increase in NaCl levels in the intestinal lumen.
- CT (A subunit): activates adenylate cyclase: catalyses conversion of ATP to cAMP.
- cAMP: increases activity of the chloride (CFTR) transporter.
- cAMP: decreases activity of sodium (NHE2/NHE3) transporters.
- Modulation of both receptors causes elevated concentrations of Na+ and Cl– ions in small intestinal lumen which becomes hyperosmotic compared to cells lining the intestines.
- Causes osmosis of large volumes of water into the intestinal lumen from the cells and ultimately from the bloodstream.
- Water exits the body in form of diarrhoea, resulting in dehydration.

Question 10

Describe ETEC, and ETEC colonisation factors

Answer 10

Enterotoxigenic Escherichia coli (ETEC)
- Gram-negative, facultative anaerobe, motile bacillus.
- Infects human host via consumption of contaminated food/water.
- Causative agent of traveller’s (abundant watery) diarrhoea in developing countries.
- Also secretory diarrhoea (along with V.cholerae )
- Responsible for an estimated 300,000 -

500,000 deaths annually in children aged <5 years.
- Clinical features and treatment are similar to cholera (emphasis on rehydration and electrolyte replenishment).

ETEC Colonisation Factors
- Colonisation of the small intestine is essential for ETEC infection
- A variety of different structures are required for adhesion:
- Fimbriae (e.g. CFA/I)
- Pili (e.g. ECP)
- Non-fimbrial adhesins (e.g. Tia, TibA)
- Colonisation of the gut is a pre-requisite for production of toxins that are directly responsible for causing the watery diarrhea
- ETEC causes toxin-mediated disease

Question 11

Describe etec TOXINS, structure and function

Answer 11

ETEC Toxin Structure and Function
Heat labile toxin (LT)
- Similar to CT in structure (AB5) and function

Heat stable toxin (ST)
- Small cysteine-rich peptide
- Binds to receptor (guanylyl cyclase C) on surface of small intestinal epithelium: catalyses conversion of GTP to cGMP:
- cGMP: increases activity of chloride (CFTR) transporter
- cGMP: decreases activity of sodium (NHE3) transporter
- Causes Cl– secretion and Na+ absorption, leading to an increase in NaCl levels in the intestinal lumen

Question 12

Describe EHEC, symptoms and treatment

Answer 12

• Gram-negative, facultative anaerobe, motile bacillus
• Infects human host via consumption of contaminated food/water
  
  • Primary sources of EHEC outbreaks are raw or undercooked ground meat products, unpasteurised milk, and faecal contamination of vegetables or drinking water
• Causative agent of diarrhoea, haemorrhagic colitis (HC), and haemolytic uraemic syndrome (HUS) ^[anemia, thrombocytopenia, and renal failure] in developing and developed settings - a type of osmotic diarrhoea
  
  • Common cause of renal failure in young children

Symptoms:
- Diarrhoea which can be bloody

Treatment:
- Rehydration and renal-supportive therapy
- Antibiotics are not recommended due to increased risk of HUS

Question 13

Describe EHEC colonisation factors

Answer 13

• Colonisation of the large intestine is essential for EHEC infection
• A variety of different structures are required for adhesion:
  
  • Intimin and Tir
  • Long polar fimbriae (Lpf)
  • Haemorrhagic coli pilus (type IV pilus)
  • Non-fimbrial adhesins (e.g., OmpA, Efa1, Iha)
• EHEC colonisation of the gut is a pre-requisite for production of toxins which contribute to the diarrhoea

Question 14

Describe attaching/effacing lesion-mediated (aka osmotic) diarrhoea

Answer 14

• Caused by an excess amount of poorly absorbed nutrients (i.e., malabsorption) that remain in the intestinal lumen
• Examples include lactulose and magnesium
• Causes retention of water in the lumen through osmotic effects
• Attaching/Effacing (A/E) lesions:
  
  • Result from destruction of intestinal microvilli and induction of cytoskeletal rearrangements directly beneath intimately attached EHEC organisms
  • Mediated by Intimin and Tir
  • Decreases surface area for nutrient absorption, causing the intestinal contents to increase in osmolarity, leading to secondary osmotic diarrhoea

Question 15

Describe shiga toxin-mediated diarrhoea

Answer 15

• Shiga toxins (Stx) 1 and 2:
  
  • Antigenically distinct AB5 toxins responsible for the progression to HUS (bind to renal cells)
  • Stx A subunit inactivates host ribosomes, thereby inhibiting protein synthesis (translation)
  • Both Stx1 and Stx2 inhibit intestinal absorption of water, causing luminal fluid accumulation which exits the body as diarrhoea

Question 16

Describe clostridioides difficile

Answer 16

• Gram-positive bacillus, obligate anaerobe, spore-forming
• Part of the normal gut flora
• Leading cause of healthcare-associated infectious diarrhoea, with spectrum of disease ranging from:
  
  • Uncomplicated diarrhoea, inflammatory type
  • Systemic toxic effects leading to significant colonic inflammation, sepsis, and death
• A hospital “superbug” due to the difficulties in treatment and the propensity to spread in nosocomial settings
• Economic burden of C. difficile infection estimated at $800 million annually in the US

Question 17

Describe C diff infection

Answer 17

• Most important risk factor for C. difficile infection (CDI) is the recent administration of broad-spectrum antibiotics which cause gut dysbiosis
• CDI can also occur when environmental spores are ingested and then reach the large intestine
  
  • Spores comprised of several heat/chemical-resistant layers, allowing them to survive acidic environment of the stomach
  • Bile salts within the small intestine trigger germination of spores into vegetative cells in the large intestine

Question 18

Describe C diff transmission

Answer 18

• Stopping transmission of C. difficile to other individuals is important and challenging.
  
  • C. difficile can easily spread by direct or indirect exposure to a patient or the patient’s environment:
    
    • Exposure to contaminated care equipment or high-touch surfaces in patients’ room/bathroom
    • Transfer of bacterial spores to patients via hands of healthcare workers
  • Oral vancomycin and metronidazole are used as treatment, however these antibiotics do not kill spores.
  • Bleach should be used to clean contaminated surfaces as normal cleaning products may be ineffective against spores.
  • Hand-washing preferred over handrubs for hygiene

Question 19

Describe C diff clinical features and treatment

Answer 19

Clinical features:
- Diarrhoea (can range from mild self-limiting to severe haematochezia, abdominal cramps, and abdominal pain)
- Pseudomembranous colitis (inflammation of the colon)- so named because C diff overgrowth forms layers resembling membranes
- Toxic megacolon (acute toxic colitis with dilation of colon) which can result in perforation of the colon, sepsis, and death

Treatments:
- Antibiotics including vancomycin (oral), metronidazole (oral or IV), fidaxomicin (oral)
- Faecal microbiota transplant (FMT) – disease remission in up to 92% of cases
- Surgical removal of colon (for severe toxic megacolon)

Question 20

Describe C diff colonisation factors

Answer 20

• Colonisation (and overgrowth) within the large intestine is essential for C. difficile infection
• A variety of different structures are required for adhesion:
  
  • S-layer proteins
  • Cell wall proteins (e.g., Cwp66)
  • Fbp68 fibronectin-binding protein
  • FliC-FliD components of flagella
• Colonisation of the colon is a pre-requisite for production of toxins that are directly and indirectly responsible for causing the (inflammatory) diarrhoea
  
  • Inflammatory diarrhoea is characterised by bloody stools, small-volume mucous stools, and fever

Question 21

Describe C diff toxins

Answer 21

Toxin A (TcdA)
- 308 kDa, binds to receptor (glycoprotein gp96) on the apical surface of colonic epithelial cells
- Causes direct alterations in barrier function

Toxin B (TcdB)
- 270 kDa, gains access to basolateral surface of colonic epithelial cells after tight junction disruption, binding to an unidentified receptor

Binary toxin (CDT)
- 147 kDa, actin-specific ADP ribosyltransferase that destabilises the cytoskeleton and potentiates toxicity of TcdA and TcdB, contributing to the severity of infection

• ## TdcA/B can be detected by PCR in stool, part of diagnostic toolkit for C. diff

C. difficile TcdA and TcdB Toxins
- Potent cytotoxic enzymes that specifically glycosylate small GTPase protein Rho, leading to disruption of cytoskeletal integrity and cytotoxicity.
- Trigger an extensive inflammatory cascade resulting in damage to host tissue and release of inflammatory exudative fluid into the colonic lumen (appears mucous-y due to presence of WCCs).
- Activate enteric nerves and (Toxin A) promote the release of neuropeptides, which elicit Cl– secretion from intestinal epithelial cells (therefore diarrhoea also has a secretory component)

Question 22

Describe **TcdA/TcdB-mediated inflammation

Answer 22

• TcdA and B are potent cytotoxic enzymes that specfically glycosylate small GTPase RHo, leading to disruption fo cytoskeletal integrity and cytotoxocoty
• triggers an extensive inflammatory cascade resulting in damage to host tissue and release inflammatory exudative fluid into the colonic lumen
• activate enteric nerves and toxin A promotes release of neuropeptides, which elicit Cl- secretion from intestinal epithelial cells
  
  • hence diarrhoea has a secretory component but it is by and large inflammatory

Question 23

Describe the Pathogenesis of C. difficile-associated Diarrhoea

Answer 23

• Toxin-induced inflammation damages tight junctions and the IEC barrier resulting in fluid exudation (inflammatory diarrhoea)
• Part of the inflammatory response includes neutrophils which release an adenosine precursor that activates CFTR thereby promoting Cl– secretion (secretory diarrhoea)
• Continual destruction of intestinal epithelium from inflammation results in:
  
  • Exudation of blood and serum into the lumen (bloody diarrhoea)- note may not be so bloody in mild disease
  • Destruction of absorptive epithelium (osmotic diarrhoea)