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MICI 1100Health Sciences Microbiology
Course Coordinator: Dr David Haldane Rm 326 Mackenzie
Building, QE II [email protected]
Welcome to
Objectives of the Course• To have an appreciation of the development of microbiology relating
to infection.• To understand the structure and physiology of microorganisms of
different types.• Be able to recognize genera and important species by name.• To have an understanding of the types of infectious disease.• To have an understanding of the role of particular organisms in
infections, and how infection is caused.• Be aware of the range of organisms causing disease, and how to
distinguish groups of organisms.• To understand the sources, and routes of transmission of organisms• To have an understanding of how infectious diseases are
manifested in the host.
Objectives• To understand the nature and role of the immune system• To know the role of immunization in the prevention of infection.• To have an understanding of the range and principle mode of action
of antimicrobial agents.• To have an understanding of the means by which organisms are
resistant to antimicrobials.• To have an understanding of the principles of environmental control
of organisms. • To have an understanding of the principles of infection control.• To be able to provide appropriate specimens and understand
laboratory results for microbiology.• To have an awareness of the laboratory techniques used in the
diagnosis of infectious disease.
Milestones in Microbiology
• Ancient and Medieval Times – microorganisms were unknown and their effects (e.g. plagues) were attributed to Divine judgement, magic, or sorcery.– 1674 - Anton van Leeuwenhoek observes
microorganisms - "animalcules" - and reports them to the British Royal Society.
– 1798 - Jenner uses the first vaccine and begins a process that will lead to the eradication of smallpox in the 1970s.
Edward Jenner (1749 - 1823)
Anthon van Leeuwenhoek (1632-1723)
Milestones (cont’d)– 17th-19th - The theory of spontaneous generation
(that organisms were generated from rotting organic material) was slowly disproved, a process which was finally completed by Pasteur and Tyndall.
– 1850 – Semelweiss shows the value of hygiene – 1860s - Pasteur furthers the germ theory of disease by
his work on silkworms, and develops pasteurization.– 1870s - Lister uses "antisepsis" to control surgical
infections.– 1876 - Koch demonstrates that anthrax is caused by
Bacillus anthracis.
Milestones in Microbiology (cont’d)
– 1881 – Lina Hesse suggests agar to solidify growth media for bacteria.
– 1880-1900 - “The Golden Age of Microbiology” - many pathogens first identified.
– 1940s - Development of antibiotics begins.– 1940s–present - Widespread use of immunization
leads to huge reductions in illness and death caused by many previously common infections, e.g. measles, diphtheria.
– 1980s - Development of molecular techniques for diagnosis and engineering begins.
Koch's postulates - to establish if an organism is the cause of a disease
• The same organism must be found in all cases of a given disease.
• The organism must be isolated and grown in pure culture.
• Organisms from the pure culture must reproduce the disease when inoculated into a healthy susceptible animal.
• The organism must then again be isolated from the experimentally infected animal.
Organisms - Morphology (shapes)
• Cocci– Streptococci (Strepto - chain)
– Staphylococci (Staphylo - grapes)
• Rods (“bacilli”) – very short rods - coccobacilli
– curved rods - vibrio
– spiral rods - spirochaetes
• Filaments with branching – actinomycetes
Staphylococci
Streptococci
Rods
Vibrio
Bacterial Morphology
Spirochaetes Cocco-bacilli
Branching FilamentsBranching Rods
More Bacterial Morphology
Structures of Bacteria
• Appendages - flagella- fimbriae- pili
• Surface and cell wall - capsule - cell wall
- cell membrane• Cytoplasm - Bacterial chromosome
- Plasmids- Ribosomes- Inclusions
• Other structures - Endospores
Appendages - project from the cell
• Flagella
– Long slender, structures made of protein
– Whip like structures
– Enable bacteria to move by rotating like a propeller
– Can only be seen using special stains or electron microscopy
– Can be single (monotrichous), or multiple, in tufts or around the cell (peritrichous).
Flagella - Arrangements
Appendages - project from the cell (cont’d)
• Fimbriae – Shorter, thinner filaments made of protein– Enable bacteria to attach to substances
• Pili – Similar to fimbriae in structure– Involved in transfer of DNA between bacteria
Appendages to Bacterial Cells
Surface and Cell Wall
• Capsule– Material that is secreted by bacteria and covers the exterior of
the cell– Often polysaccharide– May be a thick layer; slime coating
• Cell Wall– Differs from animal cells, or fungi– A strong layer made of peptidoglycan– Maintains cell shape and integrity– A principle target for antibiotic action– Stains using the Gram stain. Differs for Gram positive vs Gram
negative organisms
Surface and Cell Wall (cont’d)
• Gram Positive– Thick peptidoglycan layer– No outer membrane
• Gram Negative– Outer membrane– Thin peptidoglycan layer– Space between membranes is periplasmic
space
Surface and Cell Wall (cont’d)
• Cell Membrane– Lipid bilayer with proteins– Controls the entrance and exit of substances from the
cell– Contains enzymes involved in cell wall production,
cellular metabolism, and production of some extra-cellular materials
– In gram negatives, it contains endotoxin• Cytoplasm
– Liquid containing a variety of substances– It is where metabolism occurs
Surface and Cell Wall (cont’d)
• Ribosomes– Made of RNA and protein– Structures where proteins are made– Two subunits. Bacterial ribosomes are
different from ribosomes in animal or plant cells (eukaryotic cells)
• Bacterial Chromosome– Made of DNA– A single long circular molecular of DNA– Not separated from cytoplasm (as in animal or plant
cells which have nuclei)
Surface and Cell Wall (cont’d)
• Plasmids– Small, circular pieces of DNA– Separate from the chromosome– Can be transferred between bacteria– May carry genes for antibiotic resistance
• Inclusions– Granules in the cytoplasm– May act as storage of various substances
Other Structures
• Endospores ("spore")– Environmentally tough, dormant form– Develop in cytoplasm of bacteria– Do not grow or divide– Can remain viable for long periods– Only formed by certain genera of bacteria– Germinates to form a new cell
Bacterial Taxonomy
• How are bacteria organized and classified– Domains
• Cells lacking nuclei (prokaryotes) vs cells with nuclei (eukaryotes)
– Kingdoms*:• Animals• Plants• Fungi• Protista• Monera - the prokaryotic organisms
• (*Note: Different systems are used; this one is convenient)
Bacterial Taxonomy (cont’d)
• Classification: KingdomPhylumClassOrderFamily Used mostGenus frequently inSpecies clinical practise
Bacterial Taxonomy ( cont’d)
• Characteristics used to classify organisms
– Traditional
• Size, shape, gram reaction, need for O2
• Ability to metabolize sugars
• Metabolic end products
– Supplemented by
• Comparison of 100-300 characteristics
• Nucleic acid sequence of ribosomal RNA
General Groupings used in Taxonomy
• Aerobic (grows in air), obligate if must have O2. Capnophilic if needs CO2.
• Facultative anaerobe (grows in air, and can grow without oxygen).
• Anaerobe (grows without oxygen, and most species do not grow well in air as O2 is toxic for them).
• Microaerophilic (grows in a low concentration of oxygen, but not in its absence or in air).
Staining Organisms
• Needed to allow us to see the organisms using light microscopy
• Organisms are killed in the process
• Simple stains– stain is applied and colours the organism
e.g. methylene blue
Complex Stains
• stains may be combined which stain different structures different colours. e.g. giemsa stains malarial parasites nucleus red and cytoplasm blue
• stains may be applied in sequence with a step to remove stain in between. e.g. gram stain - a key stain in microbiology!!
The Gram Stain
• Developed by Christian Gram in the 19th Century
• He found that a stain could be washed out of some organisms much more easily than others
• Technique allows differentiation of many bacteria into 2 groups: gram positive and gram negative – corresponding to cell wall type.
• Continues to be used extensively and is important!
Method for Gram Stain
• Crystal violet – stains all the bacteria dark purple
• Iodine – binds to crystal violet and fixes it (acts as a mordant)
• Alcohol/Acetone washes out the stain from gram negative bacteria
• (Gram originally stopped here, so that organisms that stained purple were “positive” because they could be seen; subsequently the fourth step was added so that both the positive and the negative organisms could be seen.)
• Safranin stains the gram negative bacteria pink.
a
Acid Fast Stain• Some bacteria cannot be stained by the gram stain
because of lipids in the cell walls. (e.g. Mycobacterium tuberculosis, the tuberculosis bacterium) These bacteria may be stained by an “acid fast method”.
• involves: - staining with a strong red stain (to “force” the stain into the cell)
• washing out the stain with a mixture of acid and alcohol
• restaining (“counterstaining”) with a blue or green stain.
• Acid Fast organisms are Red. These are sometimes called AFB (acid fast bacilli).
• Other organisms are the colour of the counter stain (blue or green).
Bacterial Growth Requirements and Metabolism
Requirements for Bacterial Growth
• Carbon source• Nitrogen source• Essential nutrients• Temperature• Atmosphere• Inorganic ions, iron• pH• Water
Requirements for Bacterial Growth (cont’d)
• Carbon Sources– Simple carbohydrates, sugars, proteins
– Some organisms can fix CO2
• Nitrogen Sources– Protein, amino acid, peptides– Nitrates, ammonium salts
– Some organisms can fix N2
Requirements for Bacterial Growth (cont'd)
• Essential Nutrients
– Bacteria vary in their requirements
• Some can synthesize all their needs
• Others need complex organic molecules, blood, vitamins to grow. These are called fastidious.
• Temperature
– Bacteria (like humans) grow best at certain temperatures.
– Mesophiles grow best between 20-40°C. Other types are best adapted to growing below 15, or above 40-45.
– Human pathogens are usually mesophiles.
Requirements for Bacterial Growth (cont'd)
• Oxygen– Acts as a final electron accepter in aerobic organisms.
– The superoxide radical (O2-) is toxic and must be
rendered safe for cells to survive. Anaerobic organisms lack the means to detoxify O2
-.
• Iron– Required for enzyme action.– Fe3+ is insoluble.– Bacteria produce siderophores, which bind to Fe3+
and make it possible to import it.
Requirements for Bacterial Growth (cont’d)
• pH– Most organisms prefer neutral conditions.– Bacteria tend to die in acidic conditions (pH <6).
• Water– Bacteria require soluble nutrients for diffusion into the
cell.– Growth is inhibited in strong solutions.– Bacteria with defective cell walls burst in very weak
solutions.
Growth of Bacteria
• Bacteria multiply by binary fission (a single cell separates to form two new cells of equal size).
• The rate of growth is limited by:– the availability of nutrients– temperature– ability to remove toxic products
• The time required to divide is called the generation time.– for most organisms, it is measured in minutes
Phases of Growth of Microbial Populations
• Lag phase– Adaption to environment– Active synthesis of enzymes and other
constituents
• Log (i.e. logarithmic) phase– Rapid reproduction– Antibiotics most active
Phases of Growth of Microbial Populations (cont’d)
• Stationary Phase– Rate of reproduction equals rate of cell death– Nutrients depleted– Toxic metabolites accumulate
• Death Phase– Death rate exceeds reproduction
Phases of Bacterial Growth
Metabolism
• Anabolism– building organic molecules using small
molecules + energy
• Catabolism– breakdown of chemical nutrients with release
of energy
• Cells store energy as adenosine triphosphate (ATP) as substrates are oxidized
ATP
Metabolism (cont'd)
• Anabolism– Energy consuming process of building cell
components.– Protein synthesis by polymerization of amino
acids.– Glycogen and cell wall by polymerization of
glucose.– Lipids synthesis.– Nucleic acid synthesis.
Starch
Importation of Nutrients
• Active Transport - enzymes move substrate into the cell, requiring energy.
– Concentration inside the cell higher than outside
– No modification of substrate
• Group Translocation - enzymes modify a substance as it comes into the cell.
– Diffusion of altered substrate is reduced
– Energy required
Importation of Nutrients (cont’d)
• Facilitated Diffusion - enzymes aid diffusion but no energy required.– No modification of substrate– Concentration does not exceed exterior conc.
• Gycolysis - glucose is broken down to pyruvic acid, the pyruvic acid is further broken down, and the products differ for different bacteria, but include organic acids and alcohols.
Glycolysis
• Krebs cycle (also called tricarboxylic acid cycle, citric acid cycle)– pyruvate is degraded
to CO2 and H2O.
– Only used in aerobic organisms
– Results in much more energy production
Respiration
Catabolism
• Respiration– electrons pass to
O2 eventually (oxidative phosphorylation)
• Fermentation– anaerobic process,
electrons are transferred to form other organic compounds, e.g. ethanol
Other Catabolism
• lipase Lipids glycerol glycolysis fatty acids oxidized
• protease Proteins amino acids protein synthesis or further breakdown
Sterilization and Disinfection
Disinfection
• Disinfection using Chemicals.• Antiseptics - "disinfectants" that can be used on
skin.• Disinfectant - usually used on inanimate objects.
– May kill bacteria (bactericide) or prevent growth (bacteriostatic agent).
• Pasteurization
• Preservation - drying, osmotic methods, etc.
Disinfection (cont’d)
• Factors important in disinfectant activity:
– Disinfectant concentration
– Time of exposure
– Number and type of microbes present
– Nature of material to be disinfected
• Mode of action
– Disruption of cell membrane (e.g. detergents).
– Denaturation of proteins (e.g. alcohol).
Examples of Disinfectants
• Phenol based - disrupt cell membranes and precipitate proteins.– As phenol is toxic, chemically altered (“substituted”)
phenols –phenolics- are used.– Cresol - similar action to phenol. e.g. Lysol.
• Biguanides – disrupt plasma membrane. Nontoxic.• e.g. chlorhexidine used for skin disinfection.
• Alcohols - denature proteins.– 70% is more effective than 100%– Requires adequate time for activity
Examples of Disinfectants (cont’d)
• Halogens (fluorine, chlorine, iodine) - acts by oxidation of enzymes.– Hypochlorite (“javex”) is commonly used– Inactivated by organic material– Activity of preparations drops after opening
• Quarternary ammonium compounds - possibly disrupt membranes– Often combined with detergents– Commonly used for environmental cleaning
Examples of Disinfectants (cont’d)
• Detergents - disrupt cell membranes.
• Heavy metals. (e.g. copper, lead)
“High Level Disinfectants”
• Substances able to kill spores, tubercle bacilli, and viruses given enough time.– Examples
• Glutaraldehyde• Formaldehyde
Sterilization
• Elimination of viable organisms.
• Used for substances/devices to be inoculated into or to enter patients.
Methods
• Heat– moist (autoclaving)– dry (oven, less effective)
• Gas– ethylene oxide
• Oxidizing agents
– ozone, H2O2
• Irradiation• Filtration (does not eliminate viruses)
Autoclaving
• Moist heat (steam) at increased pressure for a defined time.
• Can be used for most items (e.g. surgical instruments, fabrics, etc.).
• Ability to kill spores should be checked weekly.
Gas
• Used for objects damaged by heat or radiation.
• Requires aeration step after sterilization
Radiation
• Used in industry for plastic objects, fluids, etc.
Bacterial Pathogenicity Virulence Factors and
Genetics
Microbial Ecology
• Relationships between host and microbes.– Commensal - Microbe received benefit, but
there is no harm to the host.– Opportunist - Microbe received benefit, and is
able to cause disease if host defenses are weakened.
– Pathogenicity - The ability of an organism to cause disease.
Microbial Ecology (cont’d)
– Virulence - The extent to which an organism can cause severe disease.
– Normal Flora - The community of organisms that normally exist on a body surface, the constituents vary according to the site.
Transmission of Infection
• Sources may be – from the normal flora– from other sources
• Other sources:– people– animals (direct or via food)– environment– vectors and fomites
Transmission of Infection (cont’d)
• Vector: a small organism (e.g. insect) that transmits an infectious agent.
• Fomite: an inanimate object that transmits infection when contaminated. e.g. doorknob.
• For further details, see the Infection Control lecture.
Virulence Factors
• The properties that an organism has to enable it to cause infection.
• May enable an organism to evade host defenses.
• May improve access to the body's nutrients.
– Colonization factors, e.g. fimbriae
• Allow an organism to adhere to cells.
• Adhesions are proteins that allow organisms to stick to cells.
Virulence Factors (cont’d)
– Antiphagocytic mechanisms, e.g. capsule• Body's immune cells are unable to engulf
organisms.
– Exotoxins (toxins excreted from the bacterial cell).
• A wide variety of enzymes and toxic proteins are released.
Virulence Factors (cont’d)
• Substances that help organisms invade– Hemolysins - cause lysis of red blood cells, and
damage other body cells.
– Leukocidins - kill white blood cells.
– Hyaluronidase - breaks down connective tissue extracellular material allowing spread.
– Collagenase - breaks down collagen, a structural protein.
Virulence Factor (cont’d)
• Toxins that cause disease– Enterotoxins - attack the bowel.– Neurotoxins - inhibit normal neurological function.– Protein synthesis inhibitors - can kill cells or damage
organs, e.g. diphtheria– Superantigens - these toxins bind to macrophages
and short circuit the mechanism for stimulation of the immune system, causing a massive response and consequent damage to the body, e.g. Toxic Shock Syndrome, "Flesh eating disease".
Virulence Factors (cont’d)
• Endotoxin ("Pyrogen")– Found in the outer membrane of gram
negative organisms.– Causes fever, drop in blood pressure (shock).– Acts by binding macrophages and causing
release of active substances (cytokines).
Bacterial Genetics
• Bacteria do not have nuclei.• DNA in bacteria occurs as a single circular
molecule, and sometimes as small circular molecules (plasmids) that are independent of the chromosome but are expressed.
• DNA contains the genetic code, recorded in the sequences of the 4 bases in DNA. Special enzymes cut DNA when it has the specific base sequence for that enzyme.
Bacterial Genetics (cont’d)
• Genetic information is transferred from DNA to RNA and then expressed in the form of proteins.
• As the DNA sequence of an individual strain is unique (although parts are identical for strains in the same species or genus), it is the basis for the revolution in molecular techniques that you will hear about in a future lecture.
DNA Transfer
• Free extracellular DNA can be taken up by some bacteria and incorporated to the bacterial genome (transformation).
• Transfer of genetic material by direct contact of cells (conjugation) especially important in gram negatives.– mediated by pili– allows transfer of plasmids
DNA Transfer (cont’d)
• Genetic material is transferred via a bacterial virus (bacteriophage).– Some bacteriophages rapidly destroy infected
bacterial cells– Others combine their DNA with the host
bacteria, where it can be expressed. This process is called transduction.