MICI 1100Health Sciences Microbiology

Course Coordinator: Dr David Haldane Rm 326 Mackenzie

Building, QE II HSCDavid.Haldane@cdha.nshealth.ca

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.