MMI 133

• Prion
• Virus
• Bacteria
• Fungus
• Parasite
• Animals and Us

• 1. Bacteriology
• 2. Virology
• 3. Mycology
• 4. Parasitology
• 5. Immunology

Study of bacteria

Study of viruses

Study of fungi and yeast

Study of protozoa (amoeba etc) and worms

Study of immunity (resistance to infection)

• Bacteria
• Small unicellular organisms
• All proteins begin with a special amino acid, formylmethionine

• Fungi, plant cells, protozoa, metozoa, human cells
• All proteins begin with the amino acid methionine

• 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

• 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

• Treatment options
• Determine chance of side effects
• Understand mechanism of pathogenicity
• Know what to expect in terms of disease progression and symptoms

• 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

• 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

• 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

• 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"

Subcutaneous hemorrhaging, looks like blotches or bruising along the skin

Nickname for Neisseria meningitidis

• 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

• 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

• 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

complications

• 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)

• Rapid ID of disease
• Rapid ID of contacts
• Prophylactic antibodies
• Vaccination of susceptible individuals
• Health prevention information
• Reportable disease

• Domain
• Kingdom
• Phylum
• Class
• Order
• Family
• Genus
• Species

Round

Rod

Spiral

Cluster of round bacteria

Chains of round bacteria

Live in the gut (strepto)

2 round cells

2 rod cells

Chains of rod shaped cells

Short, oval cells; usually small in size

Like a comma, short curved rods

Loose curves, wavy shape, rigid

• Telephone cord (tight) (syphillus)
• Helical, tightly coiled but flexible

• 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)

• 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

• 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)

• 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

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)

Capsule or slime layer

Motion organelle

Internal flagella in spirochaetes

• Attachment
• sex pilus: transfer of genes

Lattice structure, Macromolecule, polymer of N-acetylmuramic acid and N-acetulglucosamine (NAMA & NAG)

• Outer membrane (G-)
• Lipid A: endotoxin (important in inflammation)
• Polysaccharide = "O" antigen

• (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

• 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)

• 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

• Endospores: resting cells, resistant to heat, drying, chemicals (function for survival)
• Both Clostridium and Bacillus species produce these endospores

• 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)

• 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

• 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

• Antibiotics that target anaerobic bacteria (like metronidazole [Flagyl] or target the Gram positive cell wall [vancomycin]
• Probiotics?
• Fecal implants?

• 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%

• Sum total of all chemical reactions within an organism
• Catabolic + Anabolic = metabolic

Chemical reactions that release energy, breakdown of organic coupounds

Chemical reactions that require energy, building of complex organic molecules from simpler compounds

• Temperature dependant, higher the temp, higher the rate.
• Problem is that high temps may kill cells (denature proteins)
• Solution: 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

• Temperature
• pH
• Saturation
• Salt concentration
• Inhibitors (mercury, fluoride)

• 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

• 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

• 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

• pyschrophiles (cold loving, -5-+15C)
• pyschrotrophs (20-30C) Listeria
• mesophiles (25-45C)
• thermophiles (45-70C)
• hyperthermophiles (hot springs, 70-110C)

• Temperature
• pH
• Osmotic Pressure
• Gas
• Medium

Metals, nutrients, etc

intracellular growth

• 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%)

• 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

• 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)

• 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.

• 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

• Foods implicated as a vehicle of infection:
• Coleslaw
• Soft unpasteurized cheeses
• turkey wieners, cold cuts
• mushrooms and prepackaged salads
• unpasteurized milk

• 5-8 usual for human pathogens (neutrophile)
• Acidophiles grow at acid pH (coal mines)
• Alkalophiles grow at alkaline pH (alkaline lakes and soils)

• 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)

• 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)

• 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

• 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)

• 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

Partial breakdown (greening) of hemoglobin in the RBC under and around the bacterial colony

Total breakdown (clearing) of hemoglobin and RBC under and around bacterial colony

No breakdown of RBCs around colony

Inhibit growth of Gram positives & contains lactose and a pH indicator to differentiate between lactose fermenting and non-fermenting Gram negatives