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Pathology Michaelmas 2013 Notes

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This is an extract of our Pathology Michaelmas 2013 document, which we sell as part of our Pathology Notes collection written by the top tier of Oxford students.

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Pathology Michaelmas 2013- Experimental and clinical evidence Info on specific pathogens is all-encompassing Antibiotics still need to be covered. Perhaps more info on general bacteriology, especially experiments?

Bacteriology- GENERAL:??

CLASSICAL

EXPERIMENT demonstrating TRANSFORMATION: mice were infected with both rough (no-capsule) strains and smooth (capsule) strains. When infected with the more virulent smooth strain, the mice died. When infected with the rough strain, the mouse survived, and this also happened when the smooth strain was heat-inactivated before infection. HOWEVER, heat inactivating the smooth strain, infecting the mouse, and then infecting with the rough strain lead to the mouse dying after a while. ?There must have been exchange (transformation) of genetic information (encoding the capsule) between the strains. The INFECTIOUS DOSE of a pathogen can give us an idea of its virulence ? e.g Shigellosis can caused by as few as 10 bacteria whereas E.coli requires between 106-108. Diversifying the flora through FAECAL TRANSPLANTS helps patients with recurrent Clostridium difficile better than antibiotics. KNOCKOUT EXPERIMENTS demonstrate the importance of CAPSULES for bacterial virulence as represented by infectious dose. Moreover, the fact that incubation of a non-capsular strain in heat-inactivated serum results in that strain surviving as well as a capsular strain shows that capsules disrupt the complement-mediated opsonisation process (thus also making them antiphagocytic).

Staphylococcus Aureus: Usually part of normal human flora. Nose is the main site of colonisation; 30%
of people are colonised at any one time. Skin also an important site, especially in hospitals.DISEASES: abscesses; various pyogenic infections: e.g endocarditis, impetigo, cellulitis, septic arthritis and osteomyelitis; food poisoning; severe pneumonia (often secondary to influenza); organ abscesses following from bacteraemia,

???scalded skin syndrome and TSS. ? PATHOGENESIS is through both causing pyogenic inflammation and through producing toxins. Typical lesion is an ABSCESS where the bacteria become inaccessible to phagocytes and opsonins and general immune surveillance through a wall of fibrin and cells. These undergo central necrosis and usually drain to the outside; however there is the risk that the organisms can DISSEMINATE into the bloodstream and seed organs- much more serious. DRAINAGE is the cornerstone of abscess treatment. Important predisposing factors are INDWELLING FOREIGN BODIES e.g. tampons (these can cause TSS) and OPEN WOUNDS. Also REDUCED HUMORAL IMMUNITY especially predisposes to infection due to the pyogenic nature of this bacterium. This might include low levels of antibodies, complement, or neutrophils. CHRONIC GRANULOMATOUS DISEASE PATIENTS are also especially prone. VIRULENCE FACTORS: - Polysaccharide capsule: 11 serotypes, is antiopsonic. However high levels of specific anticapsular antibodies are protective in that they promote opsonisation; indivduals who have already been infected however are usually not protected from subsequent infections. Teichoic acids have LPS-like immunogenic properties, and through binding to fibronectin they enable adherence to mucosal cells. Catalase: breaks down H2O2 which is important part of the neutrophil's oxidative burst killing arsenal. Distinguished from other types of staphylococci by coagulase production, which catalyses the formation of fibrin and contributes to abscess formation. Protein A normally binds to the Fc receptor of IgG, preventing the activation of complement and other humoral effector mechanisms. Enterotoxin is heat resistant and causes food poisoning characterised by watery non-bloody diarrhoea- acts as a SUPERANTIGEN within the G.I. TSST also acts as an antigen and causes toxic shock. Exfoliatin causes scalded skin syndrome- acts as a protease that cleaves the desmosomes between keratinocytes in the epidermis, separating it from the granular cell layer. Membrane damaging toxins: e.g leukocidins (many types); kill leucocytes, haemolysin (causes the beta-haemolysis). Hyaluronidase "spreading factor". Beta-lactamase production is common; thus most strains resistant to penicillin. Chemotaxis inhibitory protein of staphylocci (CHIPS) is secreted by 60% of strains- blocks neutrophil chemotaxis by binding to the formyl peptide receptor and the c5a receptor to block cognate chemotactic agonists from binding. Dlt proteins cause alanine substitutions of teichoic acid that neutralise the cell surface -ve charge that normally attracts cationic antimicrobial peptides secreted into the phagosome. The bacteria EXPLOITS ANTIBODIES targeted against its fibronectin-binding proteins (e.g. teichoic acids) as the bound IgG engages a receptor on the nearby platelet surface, causing a clustering of receptors and subsequent platelet aggregation. Then grows in thrombi, escaping the attention of neutrophils. ?
Important facet of ENDOVASCULAR INFECTIONS. Mice were infection with 15 Staph aureus strains, each with a differing degree of CATALASE ACTIVITY- a correlation between catalase activity and lethality was observed. ? Addition of EXOGENOUS CATALASE led to a further increase in lethality. Staph aureus can degrade NEUTROPHIL EXTRACELLULAR TRAPS by converting them to deoxyadenosine through the action of nuclease and adenosine synthase, triggering the caspase-3 mediated death of macrophages and their exclusion from abscesses. ? Pathogenesis has evolved to anticipate host defences and repurpose them for the destruction of the immune system.

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DLT MUTANTS are more susceptible to killing by cationic antimicrobial peptides and neutrophils in vitro. TRANSCRIPTIONAL MICROARRAY STUDIES of mRNA from different strains of Staph.aureus following ingestion by neutrophils indicates a large number of differentially activated genes such as those coding for superoxide dismutases and carotenoid pigment biosynthesis. Similar study with STREP PYOGENES (see below) shows a much smaller number- thus in latter case more inhibition of the actual phagocytosis that is important?
PROTEIN A deficient mutants are phagocytosed more efficiently in utero and show decreased virulence.

Streptococcus pyogenes: Infrequent part of the skin flora and oropharynx. Expresses a cell wall antigen which is the basis for serotyping into different lettered groups. Lacks catalase so can't tolerate aerobic conditions as well as Staph aureus can.??

DISEASES: leading bacterial cause of pharyngitis and cellulitis; important cause of impetigo; necrotizing fascilitis; scarlet fever, streptococcal toxic shock syndrome; autoimmune diseases such as rheumatic fever and acute glomerulonephritis. Is an EXOGENOUS SECONDARY INVADER, relying on viral disease or disturbances in the normal bacterial flora to invade. ? in normal healthy humans the skin is a very effective barrier in addition to the mucociliary movement, coughing and sneezing. ? host phagocytic system also very important, as unlike Staph aureus it has not yet evolved effective strategies to survive in phagosomes (SEE ABOVE FOR EVIDENCE). VIRULENCE FACTORS: M-protein- antigenic variability determines the Group A subtypes. Is strongly antiphagocytic as conserved region binds to serum factor H and prevents alternative C3 convertase activity at pathogen surface (thus recruits a protein normally involved in complement control at host cell surfaces) Antibody to this provides type-specific immunity- though there are over 80 serotypes of Group A streptococci so explains why multiple infections occur. Antigenic molecular mimicry- some epitopes on M proteins are related to host tissues- basis for cross-reactive antibodies responsible for rheumatic fever and acute glomerulonephritis (this risk of autoimmunity has prevented the use of group A streptococcal VACCINES). Also means that there is a tolerant/suppressed immune response by the host. Further mimicry is achieved by having a capsule made of hyaluronic acid; this antigenically indistinguishable from hyaluronic acid in host connective tissue. As with staph aureus this capsule is also antiphagocytic. Adhesins include lipoteichoic acids, M protein (mediates attachment to CD46), Protein F (fibronectin-binding protein). Hyaluronidase acts as a spreading factor. Streptokinase is a fibrinolysin that acts as an invasin - promotes systemic spread. Streptolysin are leukocidins and hemolysins. DNAases depolymerise the free DNA present in pus, thus reducing the viscosity of the abscess material; facilitating spread. Erythrogenic toxin is a pyrogenic exotoxin that acts as a superantigen- so called because it causes the rash associated with scarlet fever; HOWEVER it is now shown to also be associated with streptococcal TSS. Exotoxin B is a protease that rapidly destroys tissue; is produced in large amounts by strains response for NECROTIZING FASCILITIS. MUTANTS lacking STEPTOLYSINS are cleared with minimal tissue injury and inflammation.

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EVIDENCE that RHEUMATIC FEVER and streptococcal infection are linked: PROMPT TREATMENT of the sore throat reduces the incidence of rheumatic heart disease. Also, once someone has contracted rheumatic fever once they are MUCH MORE SUSCEPTIBLE to recurrences
? Why do both these Staph and Strep species have all these destructive enzymes and toxins? The reason is that they are SAPHROCYTES and THINK YOU ARE DEAD- thus want to break down your tissue for nutrients. They detect markers of death, poor perfusion, tissue necrosis, anaerobic conditions, and this triggers the production of enzymes that "eat" the flesh. Thus they HIGHLY evolved to cause disease, but they are not evolved to do it in the way that it would at first appear. Explains why they are 'happy' to reside as natural members of the flora of many individuals.
? Why have MRSA strains emerged and not the equivalent for Streptococcus pyogenes? Staph. aureus are constantly present on the skin of hosts and thus exposed to more selective pressures; Staph aureus tends to colonise and infect people with conditions that are associated with hospitals and treatments; Staph aureus has far more bacteriophages that are a potent mechanism for gene transfer (transduction); Staph aureus commonly contains and exchanges plasmids unlike Strep pyogenes so even more reason for horizontal gene transfer; Staph aureus is able to survive well in the environment, so treating the patient does not represent an end-of-the-line for the particular strain being treated, even if
/when it is removed from the patient. (Strep pyogenes is much less able to survive in the environment).

Mycobacterium tuberculosis:???

AEROBIC (i.e. they are neither Gram-positive nor Gram-negative), acid-fast bacilli (they have MYCOLIC ACID in its cell wall). Indeed it is an OBLIGATE AEROBE which explains its preference for causing disease in HIGHLY OXYGENATED TISSUES such as the upper lobes of the lung and the kidney. RESISTANT and MULTI-DRUG RESISTANT strains have been found and these are posing as an INCREASING PROBLEM worldwide; the resistance is attributed to one or more CHROMOSOMAL MUTATIONS as no plasmids have been found. Humans are the NATURAL RESEVOIR. Transmission: respiratory: breathing, coughing, sneezing. Pathogenesis: interestingly it produces NO EXOTOXIN, nor does it contain ENDOTOXIN in its cell wall. ? Its SURVIVAL STRATEGY is to instead survive and multiply within PHAGOSOMES in MACROPHAGES, preventing exposure to opsonins and other harmful humoral factors; it does this by PREVENTING FUSION of the lysosome with the phagosome (which is possible due to the MYCOLIC ACIDS in the cell wall- these are complex lipids). ? Mycolic Acids are thought to be a significant determinant of virulence in MTB. Probably, they prevent attack of the mycobacteria by cationic proteins, lysozyme, and oxygen radicals in the phagocytic granule. They also protect extracellular mycobacteria from complement deposition in serum. The PRIMARY LESION occurs in the lungs at the INITIAL SITE of infection: this consists of an ACUTE INFLAMMATORY RESPONSE - it is thus an "exudative

???lesion". This lesions and the inflamed DRAINING LYMPH NODES together are called a Ghon complex. GRANULOMATOUS LESIONS consist of a central area of giant cells containing tubercle bacilli surrounded by a zone of EPITHELIOID CELLS. ? A tubercle is a granuloma that is surrounded by FIBROUS TISSUE that has undergone central caseation necrosis: these heal by CALCIFICATION and FIBROSIS. ? The point is that the bacteria are prevented from disseminating in body, and CANNOT multiply within the tubercles because of the low pH and anoxic environment. A tubercle can ERODE into a bronchus, empty its caseous contents and thereby spread the organism to other parts of the lungs, the G.I tract if swallowed, and to other people if coughed out. ? Similarly the tubercle may invade an artery or other blood supply line. The hematogenous spread of MTB may result in EXTRAPULMONARY tuberculosis otherwise known as miliary tuberculosis. ?
The SECONDARY LESIONS caused by miliary TB can occur at almost any anatomical location, but usually involve the genitourinary system, bones, joints, lymph nodes and peritoneum. DISSEMINATION into the bloodstream can occur, enabling the bacteria to reach many internal organs and wreak havoc on health. Immunity and hypersensitivity: after RECOVERY from the primary infection, resistance to the organism is mediated by CELLULAR IMMUNITY (i.e. CD4 T cells and macrophages). 90% of infections are ASYMPTOMATIC- most important determinant of whether disease occurs is the adequacy of the cellular immunity.
? Patients who are IMMUNODEFICIENT (i.e. AIDS patients) are at much higher risk for disseminated, life-threatening tuberculosis that has escaped this cellular immunity. ? MUTATIONS in the IFN-gamma RECEPTOR gene also predispose to SEVERE tuberculosis, showing the importance of this cytokine as part of the immune response. ? Moreover, GWAS studies show that NOD2 polymorphisms are critical to outcome (same as in Crohn's disease). ? ANTI-TNFalpha drugs used for RA can cause reactivation.
? In this way, PRIOR INFECTION can be determined using a TUBERCULIN SKIN TEST, which relies on a delayed-type hypersensitivity reaction (type IV). PPD is the antigen (purified protein derivative) used. THICKENING (induration) must be observed; NOT simply REDDENING. ? A POSITIVE REST indicates previous infection by the organism, but not necessarily active disease. ? Note that this is COMPLICATED by previous BCG vaccination. Virulence factors:Special mechanisms for cell entry. The tubercle bacillus can bind directly to mannose receptors on macrophages via the cell wall-associated mannosylated glycolipid, LAM, or indirectly via certain complement receptors or Fc receptors.Intracellular growth. This is an effective means of evading the immune system. In particular, antibodies and complement are ineffective. Once MTB is phagocytosed, it can inhibit phagosome-lysosome fusion by secretion of a protein that modifies the phagosome membrane. It may remain in the phagosome or escape from the phagosome, in either case, finding a protected environment for growth in the macrophage. Detoxification of oxygen radicals. MTB interferes with the toxic effects of reactive oxygen intermediates produced in the process of phagocytosis by three mechanisms: 1) Compounds including glycolipids, sulfatides and LAM down regulate the oxidative cytotoxic mechanism. 2) Macrophage uptake via complement receptors may bypass the activation of a respiratory burst. 3) The oxidative burst may be counteracted by production of catalase and superoxide dismutase enzymes.

Antigen 85 complex. This complex is composed of a group of proteins secreted by MTB that are known to bind fibronectin. These proteins may aid in walling off the bacteria from the immune system and may facilitate tubercle formation. Slow generation time. Because of this, the immune system may not readily recognize the bacteria or may not be triggered sufficiently to eliminate them. Many other chronic disease are caused by bacteria with slow generation times, for example, slow-growing M. leprae causes leprosy, Treponema pallidum causes syphilis, and Borrelia burgdorferi causes Lyme disease. High lipid concentration in cell wall. This accounts for impermeability and resistance to antimicrobial agents, resistance to killing by acidic and alkaline compounds in both the intracellular and extracellular environment, and resistance to osmotic lysis via complement deposition and attack by lysozyme. Cord factor. Cord factor is a glycolipid found in the cell walls of mycobacteria, which causes the cells to grow in serpentine cords. It is primarily associated with virulent strains of MTB. It is known to be toxic to mammalian cells and to be an inhibitor of PMN migration. Its exact role in MTB virulence is unclear, although it has been shown to induce granulomatous reactions identical to those seen in TB.??

Treatment: MULTIDRUG THERAPY is used to prevent the emergence of drug-resistant mutants; the duration of this treatment is LONG - 6 to 9 months. The reason why this is so is because of 1) the INTRACELLULAR location MULTI-DRUG RESISTANT TB is now a SIGNIFICANT PROBLEM, and things could get worse still following the discovery of TOTALLY DRUG RESISTANT TB in India. A vaccine against MTB is available. It is called BCG, and consists of a LIVE ATTENUATED STRAIN derived from Mycobacterium bovis. This strain of Mycobacterium has REMAINED AVIRULENT for over 60 years. ? It is not 100% effective. Studies suggest a 60-80% effective rate in children. It is NOT used in the US because:
? The vaccine cannot circumvent disease reactivation in previously exposed individuals.
? The vaccine does not PREVENT INFECTION, only disease. Therefore, the ENTIRE POPULATION would have to be vaccinated if the vaccine was to be considered efficacious. (I.e. can't provide herd immunity).
? Vaccination may complicate the way the TUBERCULIN SKIN TEST is read in this country, which may otherwise be used to monitor the effectiveness of antibiotic therapy.

Escherichia coli: Gram-negative rod found both within and outside the enteric tract- reservoir includes both humans and animals. Found primarily in the colon as part of the normal gut flora.

???Basis for SEROLOGICAL TYPING of many enteric rods is the 'O antigen' portion of LPS; E.coli also have the 'H antigen' (flagellar protein) and capsular 'K antigen' and the various combinations of these result in many types of E.coli. 'O-antigen' protects the bacteria against the BILE SALTS in the gut environment. Seems to have evolved its DIFFERENT BEHAVIOURS and potential to cause disease largely by the movement of "non-core" DNA which is often organized into clusters (in pathogenicity islands and/or on plasmids). ? One way to think of these organisms is as a highly evolved, integrated, controlled core with fairly conserved physiological properties that have their BEHAVIOUR DETERMINED by their virulence determining genes). MACCONKEY'S AGAR- test for lactose fermentation- can be used in the laboratory to distinguish E.coli from the non-lactose fermenters (e.g. shigella and salmonella). DISEASES: Most common cause of UTIs and Gram-negative sepsis; important cause of neonatal meningitis; agent most frequently associated with "traveller's diarrhoea"- normally this is watery, however some strains can cause bloody diarrhoea. VIRULENCE: Pili enable the organism to adhere to jejunal and ileal cells; this enables the bacteria to then have local effects on the gut lining. What these are depends on the strain- these come in many flavours as discussed above. Disease occurs either through toxin action or inflammation. A common theme is CONTACT, ADHESION and SECRETION.
-Enterotoxigenic: heat labile toxin stimulates adenylate cyclase irreversibly and constitutively via ADP-RIBOSYLATION. cAMP then activates PKA, which phosphorylates ion transporters, causing an outpouring of fluid- WATERY DIARRHOEA. No invasion or inflammation. ? This type of E coli is a simple and good example of a "PROFESSIONAL PATHOGEN" type of behaviour- It causes diarrhoea specifically, deliberately, and directly. There is also a heat-stable toxin which affects cGMP in the same way. This behaviour is pre-encoded, and programmed to occur under the appropriate conditions, and the diarrhoea that results is directly related to the further spread of the bacterium.
-Enteropathogenic: has a similar mechanism of action to Shigella- associates with the epithelium of the large intestine through a special adhesin called intimin. It secretes the receptor, Tir, for this protein using a TYPE-III SECRETION SYSTEM directly into the host cell cytoplasm, and this subsequently gets expressed on the plasma membrane. This strategy of attachment involves rearrangement of actin in the host cell, resulting in significant DEFORMATION and PHYSICAL DAMAGE -"attachment and effacement" ? BLOODY DIARRHOEA.
-Enteroinvasive: (very similar to Shigella; no surprise because genetically Shigella is really just a sub-group of E coli) causes syndrome identical to Shigellosis- SEE BELOW.
-Enterohaemorrhagic: most well-known agent is the O157:H7 serotype. Is moderately invasive and causes bloody diarrhoea through the action of a SHIGA-LIKE TOXIN which is phage-encoded. This acts by removing an adenine base from the large ribosomal RNA found in humans, thus stopping host protein synthesis. ? Specifically acts on the lining of small blood vessels- because endothelial cells therefore constantly have to renew, this causes haemorrhage. ?
As RBCs pass through the damaged endothelial area they lyse- HEMOLYTIC ANEMIA. THROMBOCYTOPENIA occurs as platelets are consumed by clotting.

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