The stuff to fix the mess Flashcards
Define the terms aetiological diagnosis, diagnostic cytology, differential diagnosis,
Aetiological diagnosis = identifying the cause
Diagnostic cytology = the process of studying cells to identify the cause
Differential diagnosis = the process of differentiating between two or more causes of similar clinical signs
How would we go about classifying bacteria, and why is this important ?
Taxanomy
16sRNA is a structural component of the 30s small ribosome unique to bacteria this gene has a slow rate of evolution
Due to this it can be used to identify bacteria to genus and species level.
Bacteria in the same taxonomic group often stain, have similar morphology and laboratory culture conditions. Also may have the same susceptibility to antibiotics.
Name the six steps in pathogenesis invasive disease causing bacteria and fungi ?
The six steps of pathogenesis
1) enters on the host (transmission)
2) colonizes and multiples on the host
3) invades the host
4) subverts hides from the host immune system
5) multiples within the host and causes essential tissue damage
6) multiples and exits the host
What is a biofilm ?
Biofilm
Biofilms are communities of bacteria, either as a single species or several species that adhere to surfaces and and produce a profuse, protective extracellular polymer substrate / matrix (EPS)
Define Dysbiosis and L-form ?
Dysbiosis = imbalance or maladaptation of the normal colonising microbes of the body.
L-form = bacteria that have temporarily stopped producing an outer membrane
Explain the functions of bacterial structures used in colonisation of the host ?
Colonisation is the ability of bacteria to attach to cell surfaces on the host at the portal of entry
Two processes
Location of cell = flagella,
Docking
* attachment is reversable non specific
* often involves electrostatic or hydrophobic forces
* bacterial biofilms produced by bacterial communities are hydrophobic allowing microorganisms to adhere to body surfaces and protect them from the hosts protective mechanisms.
Anchoring
* permanent attachment
* ligands of bacterial adhesins will bind to their complementary receptors on host cells
* anchoring prevents the bacteria being washed out eg host cilia, mucous
* bacterial adhesins = flagella, fimbriae (common pilli), outer wall proteins, capsule and the glycocalyx which consist of polysaccharide capsules and slime
* cell receptors are usually glycoproteins
* become host and site specific = ligand receptor interaction
What is the minimum infective dose ?
This is the number of microbes required at a surface for them to invade or infect the host. Pathogenic bacteria often must reach a certain number to be infectious. The greater the infectivity of the bacterium, the lower the infective dose.
Contrast the role of bacterial colonizers in health and disease ?
Under normal circumstances bacterial species colonise body surfaces and will coexist often interacting in a complex environment. These communities exist on the skin, upper respiratory tract, gastrointestinal tract and on the external genitalia.
These microflora
* provides host nutrients
* train local immunity
* competitively exclude pathogens
* aides digestion
A disruption to normal microflora by antibiotics or changes in nutrition can encourage overgrowth of opportunistic pathogens = dysbiosis
rumen = obligate anaerobic bacteria, fungi and protozoa comprise the normal microflora
Explain the role of bacterial L forms in disease ?
Bacterial L forms
Are bacteria which after exposure to event eg antibiotics have stopped producing an outer membrane
- they are able to survive in isotonic watery environments such as the intestinal, urinary and reproductive tracts
- they may restart forming an environmentally stable outer membrane when the antibiotics are depleted.
Explain where and how bacterial biofilms are formed and their role in the pathogenesis of disease ?
Biofilms are communities of bacteria, either as a single species or several species which adhere to a surfaces and produce a profuse, protective extracellular polymer substrate/matrix (EPS)
The forming of biofilms is a multistep process
1) Bacteria loosely become adhered to surfaces through hydrophobic forces in a process called docking. This process is facilitated by rough surfaces in contact with fluids, and bacterial flagella.
2) Bacteria then become anchored (more stable adherence) to a surface and have enough nutrients to grow - forming microcolonies.
3) They produce extracellular polymer matrix / substrate EPS. This matrix nourishes embedded bacteria and protects them from destructive forces within their environment eg host immune response
4) The smaller bacterial colonies can now develop into larger protective colonies.
Qurom sensing (chemical) is used by the bacteria to communicate eg produce more EPS, upregulate or down regulate certain genes for antibiotic resistance or virulence factors.
- bacteria deeper inside the biofilm have less access to nutrients, and may become metabolically inactive. This makes them resistant to attack by cytokines or antibiotics, yet they persist as a possible source of pathogens
- during maturation of the biofilm sessile bacteria will break off. Additionally in a mature biofilm bacteria will occasionally break off due to shearing forces and become planktonic - this will only become significant clinically when large numbers of bacterium break away.
What are the therapeutic implications of a biofilm ?
Explain the different ways pathogenic bacteria co-operate with other microorganisms to cause disease and relate this to the control of disease ?
Viral - bacterial synergism (5)?
Many infectious diseases are multifactorial
1) impede muco-cillary clearance
- stimulate excessive production of mucous can block airways reducing clearance
- some viruses alter the activity and number of cilia
2) Alter surface receptors
- altering the expression of host proteins, or express their own proteins causing greater adherence of bacteria
3) Damage epithelium
- viral replication and inflammation provides a portal of entry for bacteria
4) Alter immune response
- The immune response will guard itself against an excessive response following infection with a virus, it may become refractory to a secondary bacterial infection. - diversion of immune system
5) Provide bacteria nutrients
- viruses can damage cells making iron available for bacterial growth
Describe bacterial synergism (3) ?
Although bacteria can act competitively for a resource they may also act synergistically
- facultative anaerobes and obligate anaerobes
The facultative anaerobes can be beneficial by using up all the oxygen and making the environment suitable for obligate anaerobes to survive. - Some bacteria are able to release enzymes which act to liberate fucose and sialic acid from enterocytes which may act to provide nutrients for other bacteria
- intestinal microbiota produce hydrogen sulphide which is converted to thiosulphate by the enterocytes. Neutrophils release NOS which acts to convert this to tetrathionate which salmonella is able to anaerobically respire terathionate
Describe a possible mechanism for nematode, bacteria synergism ?
Describe the different methods of bacterial invasion of tissue (3) ?
Bacterial invasion of tissue
1) Use of motility (Leptospira) uses it flagella to move through a viscous substances to invade the host.
2) External factors eg arthropods saliva, animal bites or contaminated needles
3) Invasins - spreading enzymes and cell destructive enzymes
Details on Invasins
Spreading enzymes
- digestive enzymes aid in invasion directly
- hyaluronidase - breaks down hyaluronic acid present in intestinal cement of connective tissue
- collagenase breaks down collagen a scaffold for muscle cells
- kinases converts plasmin to plasminogen preventing the formation of a blood clot
Cell destructive enzymes
- phospholipases hydrolise phospholipases in cell membranes
- lecthinases destroy lecthin in cell membranes
- haemolysins pore forming in cell membranes
Define invasin and explain how extracellular bacteria use invasins to invade and spread ?
Provide examples
Invasin = Molecules produced by bacteria that assist in the invasion of host material.
cell invasion by enzyme production
Kinases - convert plasmin to plasminogen preventing blood clotting
Phospholipases - hydrolise phospholipases in cell membranes
Describe the mechanisms whereby bacteria invade the host (2) ?
How do bacteria invade the host
The zipper mechanism
Where modest cell extensions and cytoskeletal rearrangements progressively attach to the bacterial cell surface eventually engulfing the bacterium into a vacuole.
Often the binding of a bacterium requires additional molecules. Fibronectin is the bridging molecule for phagocytosis of Straplococci and Streptococci.
Virulent bacterial strains will produce Fibronectin binding protein preventing adhesion and hence phagocytosis.
The trigger mechanism
Used by gram negative bacterium (salmonella) that utilise type three secretory system (T3SS) injector to inject an effector molecule into the host cell. The infector molecule induces actin polymerisation and remodelling of the cell cytoskeleton to engulf the bacterium.
Colistridium also uses an effector molecule which injects a effector molcule through a porin, that results in damage to the villi and allows an extension of actin out of the cell which then wraps itself around the bacterium into the cell.
What mechanisms can bacteria utilise to survive within the cell ?
Survival within the cell
Once bacteria are inside the cell the vacuole formed progressively acidifies into a mature degradative phagolysosome.
- Listeria escapes the phagolysosome to survive with the cytoplasm of the cell (must occur within 30mins to prevent destruction of the bacterium)
- bacteria can modify the environment within the phagolysosome
- bacteria may also prevent vacuole -lysosome fusion
What mechanisms can bacteria utilise to survive within the cell ?
Survival within the cell
Once bacteria are inside the cell the vacuole formed progressively acidifies into a mature degradative phagolysosome.
- Listeria escapes the phagolysosome to survive with the cytoplasm of the cell (must occur within 30mins to prevent destruction of the bacterium)
- bacteria can modify the environment within the phagolysosome
- bacteria may also prevent vacuole -lysosome fusion
Describe how bacteria are able to to evade (hide) from the hosts immune defences ?
Stealth
- Sequestion
Is the ability of bacteria to hide in an immune privileged site, a site that has the least contact with the immune system
eg. Leptospira hides in the anterior and posterior chambers of the eye
- hide within a intracellular site
- Cover onself with poorly reactive antigens
eg gram negative ploysaccharide capsule, rich in sugars which dose not stimulate the immune system coli
- molecular mimicry - mimic the surface structure of host cells
Surface antigenic variation
- change their surface proteins or carbohydrates - antigenic variation before immune destruction occurs
eg Q fever Coxiella
Describe how bacteria are able to subvert or overcome host immunity through antiphagocytic responses (5)?
Antiphagocytic responses of bacterium
1) Fibronectin binding protein produced by staphylococcus aureus, which prevents the cells from becoming attached to the host immune cells.
2) leukotoxins produced by pathogenic bacteria are aimed at neutrophils and macrophages. They act as toxins causing a porin killing the neutrophils and contributing to pus formation
(The leukotoxin of Mannheimia haemolytica produces a leukotoxin which in low doses causes neutrophils to release cytokines, but in high doses will act as a porin toxin. )
3) Enzymes produced by bacteria can reduce the efficiency of the immune response. For example catalases produced by most aerobic bacteria will counteract the oxidative burst of neutrophils by inactivating the peroxides.
4) Bind to inactivate antibodies
eg Staphylococus aureus binds to IgG preventing complement mediated opsonisation
5) Distance activation of complement by the polysaccharide chain of the gram negative endotoxin
eg. Escherichia coli which have very long polysaccharide chains
How are bacteria able to survive within an intracellular site ?
Survival of bacteria within an intracellular site
Bacteria that survive in the intracellular environment allow themselves to be phagocytosed
They evade destruction by
- not activating the macrophage
- inhibit phagosome lysosome fusion
- resist destruction with phagosome
eg. Steptococi produce murein outer cell membrane prevents destruction
- Escape using porins from phagosome
Describe Haematogenous spread ?
Many bacteria will enter the blood stream from any point eg intestinal, causing a bacteraemia and if toxins are present septicaemia.
- typically in young animals with underdeveloped immune systems or haven’t received enough immunoglobulin
- some bacteria enter the lymphatics from a localised site ie the skin then enter the bloodstream via the thoracic duct.
How does Chlamdia cause tissue destruction ?
What is an endotoxin and Exotoxin ?
Endotoxin
All gram negative bacteria have lipopolysaccharide as a component of the outer cell membrane.
- foreign antigen released by the death and lysis of large numbers of gram negative bacteria
- stimulates a hyper-inflammatory response that can be life threatening endotoxemia
- destroyed by oxidising agents
Exotoxin
Gram positive and gram negative bacteria are able to produce a number of protein based exotoxins that can cause mild to serious disease
- produced through excretion or lysis
- they are specific to the bacterial species and can be neutralised by specific antitoxin antibodies produced by the adaptive immune response
- being proteins they can be inactivated by formalin, and high temperatures
- vaccines are highly effective
Explain the functions of the different structures of the endotoxin ?
Endotoxin - lipopolysaccharide
It is composed of core sugars, “O” antigen, and lipid A
Core sugar
- Barrier to macromolecules
- In Enterobacterial ompF and ompC porins exclude the passage of hydrophobic molecules and hydrophilic molecules greater than 7.00 daltons.
- this prevents penetration of bacteria by bile salts and other toxic molecules from the GI tract.
- litle variation in these sugars between gram negative bacteria
“O” antigen
- Adherence to cell surfaces
- these long polysaccharide chains are specific to pathogenic bacterial strains and confer smoothness to bacterial strains on agar.
- impedes destruction of cell by serum components, and phagocytic cells by antigenic shift
- distance complement activation
Lipid A
- similar in all gram negative bacteria
- released by actively growing/dividing bacteria or when the cell lyses
- lipid A stimulates the immune system and is what results in a hyper inflammatory response
What are the haemodynamic effects of endotoxin ?
The effect of endotoxins on the vascular system
-Endotoxin leukotriene stimulates chemical mediators such as prostaglandins and NO (nitric) oxide. The chemical mediators act to increase capillary vasodilation increasing blood flow to the site.
- Histamine is stimulated a vasoactive amine which increases the vascular permeability. This acts to increase capillary leakage by increasing the spaces between endothelial cells, allowing protein rich fluid and even red blood cells to enter the interstitium. Resulting in haemorradges and oedema respectively.
- the loss of fluid also causes a embolisms of the capillary bed which decreases the blood flow returning to the heart. Thus the heart rate will increase but the cardiac output will decrease.
- The coagulation pathway is also stimulated when there is an increase in endothelial leakage. The release and activation of large quantities of clotting factors results in microemboli (blood clots).
- Eventually blood clotting factor is depleted, leading to disseminated intravascular coagulopathy (DIC). The animal bleeds which can be see by pinpoint petechiae (pin point bleeding sites).
Poor tissue diffusion leads to metabolic acidosis.
How dose endotoxemia result in acute respiratory distress syndrome ARDS ?
There are a number of causes of ARDS including endotoxemia
The high production of cytokines by macrophages stimulated by endotoxemia causes hyperinflammation in the lungs and increased blood flow (hypertension)
- later there is a decrease in cardiac output caused by the vascular system (hypotension)
- marginated neutrophils release their cytoplasmic granules containing proteolytic enzymes which cause damage to the endothelium and pneumocytes.
- the extravascularised proteins and surfactant will form a hyaline layer, which will impede gas exchange
This will cause cyanosis and respiratory acidosis
multiple organ failure as a result of hypoxia, heart, lungs, liver and brain
How could you go about treating endotoxemia ?
Describe DNA replication ?
What is a silent or neutral mutation ?
What is required for RT PCR ?
What is a multiplex PCR ?
What are the four advantages of RT PCR over conventional PCR ?
What is recombinant DNA technology, and the three simple steps ?
What are restriction enzymes and do bacteria use them ?
How do we use plasmids as vectors ?
What is a recombinant vaccine, subunit vaccine and DNA vaccine ?
What is a recombinant subunit vaccine ?
