MMI 133

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MMI 133
2013-01-26 20:00:12
Part one

Before quiz 1
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  1. Microbiology size
    (smallest to largest)
    • Prion
    • Virus
    • Bacteria
    • Fungus
    • Parasite
    • Animals and Us
  2. 5 major categories of Microbiology
    • 1. Bacteriology
    • 2. Virology
    • 3. Mycology
    • 4. Parasitology
    • 5. Immunology
  3. Bacteriology
    Study of bacteria
  4. Virology
    Study of viruses
  5. Mycology
    Study of fungi and yeast
  6. Parasitology
    Study of protozoa (amoeba etc) and worms
  7. Immunology
    Study of immunity (resistance to infection)
  8. Prokaryote
    • Bacteria
    • Small unicellular organisms
    • All proteins begin with a special amino acid, formylmethionine
  9. Eukaryote
    • Fungi, plant cells, protozoa, metozoa, human cells
    • All proteins begin with the amino acid methionine
  10. Prokaryotic Cells
    • Size: 0.2-2.0 micron
    • "Nucleiod" area, no discrete membrane bound nucleus
    • No membrane bound organelles
    • Simple flagella
    • Not usually plasma membrane with CHO and sterols
    • No cytoskeleton
    • Ribosomes are 70s
    • Usually single circular chromosome
    • Replicate by binary fission, no meiosis
  11. Eukaryotic Cells
    • Size: 10-100 micron
    • True nucleus with membrane and nucleoli
    • All organelle are membrane bound
    • Complex flagella if present
    • Plasma membrane with CHO an sterols
    • Cytoskeleton present
    • Ribosomes are 80s + 70s in mitochondria
    • Multiple linear chromosomes
    • Replicate by mitosis and meiosis
  12. Important t know difference between prokaryotes and eukaryotes
    • Treatment options
    • Determine chance of side effects
    • Understand mechanism of pathogenicity
    • Know what to expect in terms of disease progression and symptoms
  13. Meningitis
    • Inflammation of the meninges
    • Meninges are coverings of the brain and spinal cord (protect and enclose)
    • Usually caused by microorganisms infecting the cerebral spinal fluid
  14. Neisseria meningitidis
    • One of the causes of meningitis
    • Gram Negative diplococcus, capsule important for disease
    • >12 serotypes based on capsule carbohydrates (polysaccharides)
    • Only 4 subtypes called serotypes are responsible for most of the epidemics in the world (A,B,C,W-135)
    • Serotype A most common in Africa
    • Serotype B most common in Americas
    • Only affects humans: no animal reservoir
  15. Gram Staining
    • Crystal violet stain- enters all bacterial cells through cell wall
    • Iodine = mordant: enters through cell wall of all cells, formation of a large CV-I complex
    • Acetone-alcohol wash - decolourization: removes stain from all cells except if there is a thick peptidoglycan layer in the cell wall
    • Safranin - counterstain: Gram negative cells are colourless and need to be coloured for microscopy
  16. Pathogenicity of N. meningitidis
    • Droplet transmission (coughs, sneezes, saliva) (range 1m)
    • Attaches to mucosal membranes and invades o bloodstream
    • Contains "endotoxin" in the cell wall - causes inflammation (LOS)(lipo-oligopolysaccharide)
    • Endotoxin activates blood clotting, leads to DIC (disseminated intravascular coagulation) and hemorrhage (petichiae, purpura)
    • Evades phagocytosis (being eaten) by white blood cells due to a carbohydrate (polysaccharide) layer around the outside of the bacterial cell wall - the "capsule"
  17. Ecchymoses or purpura
    Subcutaneous hemorrhaging, looks like blotches or bruising along the skin
  18. Meningococcus (pl. meningococci)
    Nickname for Neisseria meningitidis
  19. Meningococcal meningitis
    • Bacteria in cerebrospinal fluid only
    • Can cause death if untreated: pressure to brain, influx of inflammatory cells to CSF (cerebral spinal fluid)
    • If treated early has very good prognosis
    • Signs and symptoms:
    • Headache, stiff neck, nausea, sore throat, coma, petichiae, photosensitivity
  20. Meningococcal sepsis
    • Bacteria replicate in the blood
    • Disseminated infection, can occur quickly, Gram negative sepsis, intravascular coagulation, need for amputation
    • Signs and symptoms:
    • Fever, petichiae/pupura, shock, hemorrhage, DIC - internal organ meltdown
  21. Vaccine for meningococcus
    • Two types:
    • Polysaccharide (antibodies will be made to capsule): effective against 4/15 types (A,C,Y, W135) 90% protection for 5 years
    • not used for pediatric patients <2 years of age
    • Conjugated: polysaccharide combined with a protein for increased immunogenicity - good for children < 2 and immunosuppressed individuals who can't make antibodies effeciently
  22. Sequalae
  23. What happened in AB
    • First case in Dec 1999 - outbreak lasted to June 2001: 61 cases (2 deaths)
    • Total of 56 isolates: 50/56 typed by lab as Serogroup C
    • Resulted in mass vaccination program which stopped the outbreak
    • Outcome for patients: 70.5% full recovery, 3.3% died, 6.6% amputations, 11.5% severe scars, 14.5% other sequlae (neurological problems mostly)
  24. Public Health issues from Neisseria meningitidis
    • Rapid ID of disease
    • Rapid ID of contacts
    • Prophylactic antibodies
    • Vaccination of susceptible individuals
    • Health prevention information
    • Reportable disease
  25. Classification of organisms
    • Domain
    • Kingdom
    • Phylum
    • Class
    • Order
    • Family
    • Genus
    • Species
  26. Cocci
  27. Bacilli
  28. Spiral Bacteria
  29. Staphylococci
    Cluster of round bacteria
  30. Streptococci
    Chains of round bacteria
  31. Enterococci
    Live in the gut (strepto)
  32. Diplococci
    2 round cells
  33. Diplobacilli
    2 rod cells
  34. Strepto-bacilli
    Chains of rod shaped cells
  35. Cocco-bacilli
    Short, oval cells; usually small in size
  36. Vibrio spiral bacteria
    Like a comma, short curved rods
  37. Spirillum (spirilla)
    Loose curves, wavy shape, rigid
  38. Spirochaete
    • Telephone cord (tight) (syphillus)
    • Helical, tightly coiled but flexible
  39. Usefulness of Gram stain
    • Rapid
    • Gives very useful information that is clinically relevant
    • Gives the lab a hint about further tests required to identify the organism
    • Allows us to see if there is bacteria in the specimen (don't always grow on media)
  40. Comparison of Gram negative and Gram positive organisms
    • Gram negative: Thin layer peptidoglycan
    • No teichoic acid
    • Lipopolysaccharide
    • Outer membrane and periplasm
    • Generally less susceptible to penicillin

    • Gram positive: Thick layer peptidoglycan
    • Teichoic acid present
    • No lipopolysaccharide
    • No outer membrane or periplasm
    • Generally more sysceptible to penicillin it disrupts the peptidoglycan
  41. Important exceptions to Gram staining
    • Mycobacterium sp. eg. M. tuberculosis (cell wall waxy, no penetration of Gram stain)
    • Mycoplasma sp. eg. M. pneumonia (no cell wall)
    • Chlamydia sp. eg. C. trachomatis (ebs to small to be seen and rbs intracellular)
  42. Ziehl-Neelsen or "acid fast" stain
    • Used for Mycobacterium, Nocardia, and a few other organisms like a parasite called Cryptosporidium
    • All bacteria haves waxes in their cell walls. Carbol fuchsin is added to smear and heated - this enhances the penetration of the dye through the wax. Decolorization is done by exposing the smear to acid alcohol - this removes the red stain from the organisms that are not acid-fast.  The counter stain, methylene blue, then colors the background so that it is easier to see the red-stained organisms
  43. Negative staining for capsules
    Some organisms have a polysaccharide capsule surrounding the cell walls, which are important for virulence. These capsules cannot be stained, so we use a negative stain, where the background is stained and the organism appears as a particle with a halo around the cell where the stain has been excluded (eg. India ink staining for a yeast called Cryptococcus)
  44. Glycocalyx
    Capsule or slime layer
  45. Flagella
    Motion organelle
  46. Axial filament
    Internal flagella in spirochaetes
  47. Fimbriae & Pili
    • Attachment
    • sex pilus: transfer of genes
  48. Peptidoglycan
    Lattice structure, Macromolecule, polymer of N-acetylmuramic acid and N-acetulglucosamine (NAMA & NAG)
  49. Lipopolysaccharide (LPS)
    • Outer membrane (G-)
    • Lipid A: endotoxin (important in inflammation)
    • Polysaccharide = "O" antigen
  50. Teichoic and Lipoteichoic acids
    • (only gram +)
    • Attached to peptidoglycan layer, project outwards and cause cells to have a negative charge: may function in attachment of bacteria to host cells
  51. Cytoplasm (plasma membrane)
    • Surrounds the cytoplasm of the cell
    • Site of energy production in prokaryotes
    • Prokaryotes have phospholipids cytoplasmic membranes
    • Eukaryotes = some phospholipids, but also sterols and carbohydrates (human membranes have cholesterol, fungal membranes have ergosterol)
  52. Cytoplasm of Bacteria
    • Contains:
    • Nuclear area (nucleoid): chromosome (usually 1 in bacteria, 46 in human cells)
    • Plasmids: sometimes
    • Ribosomes: site of protein synthesis, 70 S
    • Inclusions: reserve deposits of nutrients
  53. Bacterial Endospores
    • Endospores: resting cells, resistant to heat, drying, chemicals (function for survival)
    • Both Clostridium and Bacillus species produce these endospores
  54. Clostridium difficile
    How to get this
    • Present as normal flora in 3 % of healthy adults
    • Most often acquired nosocomially (in the hospital)
    • Earlier antibiotic treatment eliminated other bacteria but not the Clostridia
    • (Note, alcohol hand scrubs do not kill in inactivate spores)
  55. Clostridium difficile
    What is it?
    • Strict anaerobe, Gram positive bacillus
    • Bacteria produce Exotoxins A + B
    • Damage to intestinal mucosa
    • Toxin A = enterotoxin (fluid accumulation)
    • Toxin B = cytotoxin (loss of cell structure, cell death ensues)
    • Spore forming
    • Spores survive months I environment
  56. Clostridium difficile

    • Range from mild diarrhea (C. difficile associated diarrhea or CDAD) to pseudomembranous colitis (PMS)
    • Complications due to infection are:
    • Dehydration
    • AAC (antibiotic associated colitis)
    • PMC (pseudomembranous colitis)
    • Toxic megacolon
    • Death
    • ** Rule of thumb, children <2 years never have C. difficile infections
  57. Clostridium difficile

    • Antibiotics that target anaerobic bacteria (like metronidazole [Flagyl] or target the Gram positive cell wall [vancomycin]
    • Probiotics?
    • Fecal implants?
  58. Clostridium difficile
    in Quebec
    • Change to alcohol based hand sanitizers to replace hand washing
    • Outbreak of nosocomial C. difficile in hospitals and institutions
    • -no surveillance done originally
    • -1703 patients found in 2004
    • -Antibiotics associates were fluoroquinoloes and cephalosporins
    • -death rate 6.9%
  59. Metabolism
    • Sum total of all chemical reactions within an organism
    • Catabolic + Anabolic = metabolic
  60. Catabolism
    Chemical reactions that release energy, breakdown of organic coupounds
  61. Anabolism
    Chemical reactions that require energy, building of complex organic molecules from simpler compounds
  62. Reaction Rates
    • Temperature dependant, higher the temp, higher the rate.
    • Problem is that high temps may kill cells (denature proteins)
    • Solution: enzymes
  63. Enzymes
    • Large protein molecules (biological catalysts)
    • Reactions are at least 100,000,000X faster using enzymes
    • Substrate specific
    • May need cofactors (metal ions like zinc or magnesium)
    • Recyclable, unchanged during reaction
  64. Enzymes sensitive to:
    • Temperature
    • pH
    • Saturation
    • Salt concentration
    • Inhibitors (mercury, fluoride)
  65. Energy Production
    • Carbohydrates typically #1 source (glucose)
    • Two processes by which glucose is used
    • a. cellular respiration (aerobic, anaerobic)
    • b. fermentation
    • both processes use glycolysis where final product is pyruvate
  66. 2 types of respiration
    • Aerobic Respiration: produces more energy (ATP) than anaerobic processes, so these aerobes usually can grow faster (36-38 ATP)
    • -bacteria that need O2 to grow are obligate aerobes

    • Anaerobic Respiration: less ATP is produced (2-4)
    • bacteria usually grow slower
    • bacteria that can only grow without O2 are called obligate anaerobes
  67. Fermentation
    • Important biological process
    • Does not need oxygen but can occur in the presence of oxygen
    • Produces very small amounts of ATP because the energy in not released in the end product, it remains in the bonds
    • Lactic acid, CO2 and alcohol important products
  68. Temperature: physical factor affecting Microbial Growth
    • pyschrophiles (cold loving, -5-+15C)
    • pyschrotrophs (20-30C) Listeria
    • mesophiles (25-45C)
    • thermophiles (45-70C)
    • hyperthermophiles (hot springs, 70-110C)
  69. Physical factors that affect Microbial growth
    • Temperature
    • pH
    • Osmotic Pressure
    • Gas
    • Medium
  70. Chemical Factors that affect Microbial Growth
    Metals, nutrients, etc
  71. Environmental factors that affect Microbial Growth
    intracellular growth
  72. Listeria monocytogenes
    • Gram positive, non-spore forming
    • Facultatively anaerobic, motile
    • Grow well on complex media (BAP)
    • Grow over wide temp. range (3-42C)
    • Grow over wide pH range (<5.5 - 9.5)
    • Grow in high concentrations of NaCl (up to 10%)
  73. Listeria Growth
    • Normal Habitat
    • Widely distributed in nature, soil is probably the natural reservoir
    • Animal intestine through contaminated feed
    • Hardy, able to survive in low temperatures, high acidity and salt concentrations
  74. Clinical Presentation of Listeriosis
    • Causes disease in man and animals
    • In animals: CNS infections, goats, cattle, "circling disease"; septic abortion
    • In man: food borne disease; CNS infections; miscarriage (spontaneous abortion)
  75. Pathogenesis of Listeria
    • Ingestion of contaminated food
    • invasion of bacteria through gastric epithelial barrier (bacteria have an "invasion" which is used to gather and rearrange actin onto the "tail" of the bacterium. This tail then propels bacteria from one cell to another like a rocket)
    • Grows in splenic and hepatic macrophages, destroys phagosomes with a hemolysin called listeriolysin and can move freely in cytoplasm
    • Intracellular growth allows it to hide from white blood cells.
  76. Clinical infection of Listeria
    • Meningitis, encephalitis, septicemia: incubation period may be as long as 2 months - elderly and immunocompromised have increased risk for disease.
    • Mortality of CNS infectios 20-50%
    • Difficult to diagnose meningitis which is a chronic type and takes a long time to develop
    • Pregnant women: special case: influenza-like bacteremic illness that can result in abortion or still-birth
  77. Transmission of listeria
    • Foods implicated as a vehicle of infection:
    • Coleslaw
    • Soft unpasteurized cheeses
    • turkey wieners, cold cuts
    • mushrooms and prepackaged salads
    • unpasteurized milk
  78. pH, physical factor affecting microbial growth
    • 5-8 usual for human pathogens (neutrophile)
    • Acidophiles grow at acid pH (coal mines)
    • Alkalophiles grow at alkaline pH (alkaline lakes and soils)
  79. Osmotic pressure, physical factor affecting Microbial growth
    • Nature wants to be balanced. Thus live in an environment with similar concentration as self. 
    • Some bacteria have developed a tolerance to hypertonic environments:
    • Obligate halophiles need high salt concentrations (Dead Sea halophiles need 30% salt for growth)
    • Facultative halophiles tolerate salt, usually 2%, sometimes as high as 15% (often human pathogens)
  80. Gaseous Requirements, physical factor affecting Microbial growth
    • Test this by growing bacteria in test tubes to see where they grow.
    • Obligate aerobes (need 02)
    • Facultative anaerobes (can live without O2)
    • Obligate anaerobes (can't live with O2)
    • Aerotolerant anaerobes (can live with some O2)
    • Microaerophiles (aerobic, require oxygen but grow in oxygen concentrations lower than in air)
  81. Biofilms
    • Bacteria like to live in communities
    • >85% of human infection involves biofilms
    • Slime matrix with polysaccharides, proteins, DNA, bacteria attached to surfaces in the body or on foreign matter in the body
    • White blood cells cannot penetrate
    • Antibiotics ineffective against the bacteria living in biofilms
  82. Microbial Culture: How to grow bacteria
    • Need sterile nutrient material (solid, semi-solid or liquid)
    • AGAR: complex polysaccharide from algae for solid culture matrix (looks like gelatin) (often add animal blood (sheep, horse) for nutrients)
    • Bacteria grow in "colonies" - piles of bacteria where the original bacteria was placed on the surface of the agar plate
    • Different formulations used for culture of different bacteria
    • Differential media: eg. blood agar, hemolytic reaction
    • Selective media - prevent some species from growing and induce others to grow (usually add chemicals or antibiotics)
  83. Differential Media example
    • Hemolysis (breakdown) of sheep red blood cells that are mixed with agar to form a growth substrate for bacteria; -used for presumptive bacterial identification of some bacterial species, eg streptococci
    • (alpha, beta or gamma create different colors by breaking down parts of the RBC
  84. Alpha Hemolysis
    Partial breakdown (greening) of hemoglobin in the RBC under and around the bacterial colony
  85. Beta Hemolysis
    Total breakdown (clearing) of hemoglobin and RBC under and around bacterial colony
  86. Gamma Hemolysis
    No breakdown of RBCs around colony
  87. MacConkey Plate
    Inhibit growth of Gram positives & contains lactose and a pH indicator to differentiate between lactose fermenting and non-fermenting Gram negatives