Diarrhoeal diseases - Workshop - "Lectures 2+ 3 only. Lecture 1 is covered in Worksheet answers. Flashcards
Give named example of bacterial, protozoal and viral agent of infectious diarrhoea. Outline potenital virulence mechanisms of vibrio cholerae
- Vibrio cholerea
- Cryptospordium
- Rotovirus
Cholera toxin causes activation of intracellular adenylyl cyclase, generating cAMP this causes lots of activation of the CFTR channel, drawing chloride in the lumen - to counteract this and confer a charge balance in the lumen, sodium also moves into the lumen - which of course also draws water into the lumen
it is also thought that cholera toxin loosens tight junctions
Outline pathogenesis of cryptosprodium
- oocysts ingested - sporozites hatch fromt this
- These motile sporozoites bind to the receptors on the surface of the intestinal epithelial cells and form a parasitophorous vacuole near the surface of the epithelial cell, separated from the cytoplasm by a dense layer - the vacuole protects them from phagocytosis
- Continue to undergo a series of development and changes
- oocytes are released into feces to find new host
- Because the paracites are being maintained on the epithelial lining, they can cause cell damage
- the disruption of tight cell junctions, a loss of barrier function, the release of lactate dehydrogenase, and increased rates of cell death - seem to also release various proteases that may harm cells
Outline pathogenicity of norovirus
- Infects enterocytes in the small intestine - is internalised. Inside, viroplasms are formed where viral RNA replication takes place
- Intracellular events - likely involving the viral enterotoxin NSP4, causes calcium release rfrom ER - triggering disruption of microvillar cytoskeletal network, lowered expression of brush border enzymes at the apical surgace and general inhibition of sodium co-transporters - also appears to stimulate CELL NECROSIS - all of this points to a malabsorption problem which leads to osmotic diarrhoea
- These cells will die, and NSP4 will be released which has been produced inside the infected cells
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NSP4 can act on unafffected cells, also causing these cells to have calcium release from the ER - causing disruption of tight junctions - resulting in increase paracellular permeability - this seems to be when NSP4 affects ENETROCYTES
- If NSP4 acts on the crypt cells - the effects are sightly different - whereinstead it seems to prommote cholride secretion highlighting a secretory component of the diarrhoea
- NSP4 may also stimulate the enteric nervous system - possibly also increasing secretion
Salmonella typhi is an organism that does not appear to require enterotoxins. How does it do this?
S typhi and paratyphi enter the host’s system primarily through the distal ileum. They have specialized fimbriae that adhere to the epithelium over clusters of lymphoid tissue in the ileum (Peyer patches), the main relay point for macrophages traveling from the gut into the lymphatic system. The bacteria then induce their host macrophages to attract more macrophages. [3]
S typhi has a Vi capsular antigen that masks PAMPs, avoiding neutrophil-based inflammation, while the most common paratyphi serovar, paratyphi A, does not. This may explain the greater infectivity of typhi compared with most of its cousins. [5]
Typhoidal salmonella co-opt the macrophages’ cellular machinery for their own reproduction [6] as they are carried through the mesenteric lymph nodes to the thoracic duct and the lymphatics and then through to the reticuloendothelial tissues of the liver, spleen, bone marrow, and lymph nodes. Once there, they pause and continue to multiply until some critical density is reached. Afterward, the bacteria induce macrophage apoptosis, breaking out into the bloodstream to invade the rest of the body. [4]
The bacteria then infect the gallbladder via either bacteremia or direct extension of infected bile. The result is that the organism re-enters the gastrointestinal tract in the bile and reinfects Peyer patches. Bacteria that do not reinfect the host are typically shed in the stool and are then available to infect other hosts.
How could an organism cause diarrhoeal or vomiting illness without replicating?` Name species that do this
- May produce toxins on food OUTSIDE of the body
- Bacillus Cerus, S.aureus
What properties enable bacteria to colonise the small intestine?
- Anaerobic
- Fimbriae
- Flagellum for motility
- resistance to low pH
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Name infection control precuations
- Handwashing
- Appropriate handling and storage of food (ensuring temperatures do not permit bacterial growth)
- Proper cooking of food
- Aseptic technique - [[proper cleaning and disinfection of care equipment and instruments
- follow respiratory hyegine/cough etiquette principles
Name ways to prevent the transmission of infection
- Isolation rooms
- low pressure rooms
- Appropriate shielding from infected patients - PPE
- Limit transport and movement of patients outside of the room to medically-necessary purposes. When transport or movement is necessary, cover or contain the infected or colonized areas of the patient’s body. Remove and dispose of contaminated PPE and perform hand hygiene prior to transporting patients on Contact Precautions. Don clean PPE to handle the patient at the transport location.
Explain the differences between selective media and differential media for identification of bacteria
Selective:
- Only certain organisms will grow on these - growth of other organisms will be inhibited
Differential media:
- typically make use of a dye or sorts, relying on the biochemical or enzymatic characteristics of organims - causing a colour change
Organisms can be identified by API testing (analytical profile indexing). What is this?
Series of biochemical tests. these tests are inoculated with a bacterial suspension During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents - basically only certain bacteria will cause the colour changes.
VITEK is an automated version of this.
State methods more likely to be used in future for bacteriotyping
MALDI-TOF - mass spec
PCR - also used in viruses
Whole genome sequencing
Describe 3 types of toxins produced by bacteria that cause diarrhoeal disease. Outline their mechanisms. Describe roughly what each does and give an example.
- Neurotoxins - toxin is classified in this group if available data suggest that atleast part of the secretory activity of the toxin is attributable tothe release of one or more neurotransmitters from the enteric nervous system or that the toxin alters smooth muscle activityin the intestine
- Cytotoxin - toxins produce cellular damage documented by grossfindings (e.g., intestinal hemorrhage), light-microscopic evidence of intestinal damage, or studies demonstrating cellular injury
- Secretory entertoxin - toxin stimulates net secretion in one or more intestinal models withouteither histologic evidence of intestinal damage
Neurotoxin - CLostridium botulinium, Staph Aureus
Secretory Enterotoxin group - Vibrio Cholera , Salmonella
Cytotoxin group - Shigella, E.coli 0.157
What are the classes of enteric infection? explain what each one is and give an example.
Non-inflammatory (entertoxin or adherence/superficial invasion) - this is typically secretory, cell death is not expected - Vibrio Cholerae
Inflammatory (invasion, cytotoxin) - cell death is expected - E.coli - shigella
Penetrating (penetrating) - infection that enters the bloodstream - Salomella typhi
What is meant by phage typing when referring to techniques to identify bacteria?
What bacteria is often identified by this method?
The viruses that infect bacteria are called bacteriophages (“phages” for short) and some of these can only infect a single strain of bacteria. These phages are used to identify different strains of bacteria within a single species. A culture of the strain is grown in the agar and dried. A grid is drawn on the base of the Petri dish to mark out different regions. Inoculation of each square of the grid is done by a different phage. The phage drops are allowed to dry and are incubated: The susceptible phage regions will show a circular clearing where the bacteria have been lysed, and this is used in differentiation
Salmonella.
What is molecular typing?
Techniques that allow for identification of isolates or strains below species or subspecies level, even those that are very closley related. This is done with techniques that do fragement based sequencing, whole genome sequencing, PCR and more.