HUBS 1416 Microbiology Lecture 1

THE AGENTS OF INFECTIOUS DISEASE

PATHOGENS

Ann-Louise Lettoof, 2011 School of Biomedical Sciences and Pharmacy

Microorganisms of medical importance

Bacteria - prokaryotes. Largest group of medically significant microbes with many thousands of species. Viruses. Protozoa. Algae. Fungi. Multicellular parasites.

Bacteria were the first colonisers of the earth 4 billion years ago found in every habitat on earth In soil and deep in earths crust In aqueous solutions fresh/sea water Around deep sea vents In radioactive waste

Bacteria

free-living prokaryotes.

No nucleus/nuclear membrane No membrane-bound organelles Cell membrane usually surrounded by cell wall - principal component is peptidoglycan. Many have external structures flagella, pili etc. May have a layer external to the cell wall eg. glycocalyx, capsule, slime layer

Cytoplasm contains ribosomes DNA (usually circular DNA) localised in nucleoid region and may be plasmids (small circular DNA) usually granules/vesicles nutrient reserve

Compare eukaryotic and prokaryotic cells

Bacterial cell wall

Gram-positive cell wall characterised by a thick peptidoglycan layer possess complex polysaccharides called teichoic acids.

Gram-negative cell wall possesses a thin peptidoglycan layer

bounded by an outer membrane that contains lipopolysaccharide (LPS). LPS contains endotoxin (causes toxic effects in Gram-negative infections).

Gram positive

Gram negative

Culture and Identification of Bacteria

First step is usually to establish a pure culture of bacteria in the laboratory.

In clinical setting obtain specimen from patient - this is a mixture of bacteria in most cases
Bacteria grown in liquid or solid culture medium eg agar plate.

Culture media provide nutritional factors, stable pH and isotonic environment for growth and reproduction.
Some bacteria cannot be grown in media (Mycobacterium leprae,

Chlamydia

Culture and Identification of Bacteria

Bacterial growth dependent on chemical (nutritional) and physical factors. Physical factors include temperature pH - optimum close to neutral for most bacteria Oxygen aerobic (obligate) or anaerobic (obligate, facultative), water and osmotic pressure isotonic (optimal). hypotonic (lysis), hypertonic (shrinking). Chemical factors include carbon - heterotrophic, autotrophic nitrogen phosphorus sulphur cations trace elements (cofactors) organic factors (vitamins).

Bacterial Growth

An increase in cell number (not size!)

Reproduce by binary fission doubling cell numbers with each generation

Four phases of growth: lag, exponential, stationary and decline. Exponential or logarithmic increase

A single E coli cell can give rise to over 1million cells in 8-10 hours!! Mild infection to serious infection!!

Generation time time taken to reproduce

varies from organism to organism and with the environment may be as little as 20 mins (E.coli) or several hours (Mycobacteria

Four phases of growth: lag, exponential, stationary and decline.

Pure Cultures

Liquid media may be used initially to increase the number of bacteria in the sample (blood, anaerobes) Many grow overnight (15-18 hours). Follow with culture on solid media (agar) in a Petri dish.

Presumptive Identification
Usually based on: Gram stain - most bacteria stain Gram-positive (purple) or Gramnegative (pink). Other stains - flagella, capsule, acid fast

Cell morphology Colony morphology Aerobic, anaerobic growth (or both) Specialised nutritional requirements for growth.

Gram staining of bacteria

GRAM-POSITIVE COCCI IN CHAINS (Streptococci)

GRAM-NEGATIVE RODS (Escherichia coli)

GRAM-POSITIVE COCCI IN CLUSTERS (Staphylococci)

Bacterial Cell Morphology

Three principal shapes - spheres (cocci), rods (bacilli) and spirals (spirilli) and may possess flagella. May grow in chains or clusters.

Bacterial Colony Morphology

Colony colour, shape, lustre etc differs between species

Further tests to identify bacteria

Various other techniques to further identify bacteria with greater specificity:

Serological tests - the detection of microorganisms by specific antibodies.

Molecular techniques - detect nucleic acids fragments Biochemical tests - battery of biochemical tests (metabolic products including enzymes, sugars, alcohols and acids).

Resistance or susceptibility to antibiotics.

Viruses

Sub microscopic entities Obligate intracellular parasites

Viruses are not cells They have no nucleus, organelles or cytoplasm Can only replicate inside host cells
Mostly damage cells in which they replicate causing lysis or gradual death

Extracellular phase (virion) unable to replicate capable of host to host transmission must be assembled from synthesised components. Intracellular phase existence as a replicating nucleic acid directs the synthesis of virion components using the host cell biochemical replication mechanism

Virus or viral particle - Viral DNA or RNA (viral genome) surrounded by protein shell (capsid) Lipid Envelope Nucleic Acid

Protein Capsid Virion Associated Polymerase

Polymerase enables insertion in host DNA

Spike Projections

Structure of Viruses
Virus or viral particle - intracellular infectious particle Viral DNA or RNA (viral genome) surrounded by protein shell (capsid) Nucleocapsid protein/nuclec acid complex Envelope - some viruses possess outer coat derived from host cell( lipid bilayer) non-enveloped viruses referred to as naked viruses. Virion entire mature virus particle may be enveloped or naked found outside host capable of transmission

The Viral Capsid

Made up of repeating protein units capsomeres Occur in distinctive shapes May possess distinctive antigenic structure. Involved in the attachment and entry of virion into a host cell.

Protect viral nucleic acid against nuclease enzymes.

Resistant to desiccation, pH change and detergents (naked viruses).

May be used in identification.

Viral Envelopes

Outer membranous layer surrounding the nucleocapsid. Normal host cell constituents but proteins virally encoded. Proteins may project as spikes (peplomers) from the envelope. Spikes involved in attachment of virus to host cells. Determine antigenic characteristics.

Classification of Viruses

At present separated into major groups or families based on size morphology type of nucleic acid mode of replication DNA viruses associated with human disease fall into seven families.

RNA viruses associated with human disease fall into at least 15 families.

Replication of animal viruses

Major steps in replication is the same for all viruses.

Host cell provides substrates, energy and hardware.

Any additional requirements provided by the virus. Assembly of new viral particles under control of viral genes.

Replication of animal viruses

Six principal steps: 1. Adsorption - attachment to host cell receptor. eg via spike

2. Penetration - direct, membrane fusion, phagocytosis.

3. Uncoating - nucleic acid released into cytoplasm. 4. Synthesis - copy nucleic acid many times for protein 5. Assembly in nucleus (DNA virus) or cytoplasm (RNA virus) 6. Release - naked viruses usually result in cell lysis, enveloped viruses bud.

Identification of viruses

Most viral diseases diagnosed by signs and symptoms. May need to identify the virus to determine level of infectivity and risk of transmission. Identification techniques include Reaction with immunoflourescent antibody

Serology detection of viral antigens and specific antibodies in host eg enzyme immunoassay, ELISA molecular techniques eg nucleic acid analysis cell/tissue culture methods

Growth of viruses in the laboratory

Innoculate live animals (in vivo) Isolate new viruses Study pathogenesis or oncogenesis In embryonated eggs and cell/tissue culture (in vitro). Study influenza Make vaccines from harvested virus Allergic reactions Cell culture methods commonly used innoculate human cells grown in culture observe cytopathic effects (CPE)

Production of inclusion bobies Lysis/death Change in cell behaviour - Cells can swell, have functions arrested or become detached from tissue mass Syncytia - Cells form together into a large multinucleated mass

Protozoa and Algae

Unicellular eukaryotes Mainly aquatic

Algae also possess chlorophyll (protozoa do not) Algae do not cause infectious disease but do produce toxins.
Relatively few protozoa cause human disease (Amoebae, Giardia, Cryptosporidium, Plasmodium).
Giardia attaches to small intestine burrows in and causes release of tissue fluids causes diarrhea, malaise and lots of gas

Fungi

Diverse group of unicellular and multicellular eukaryotes. Unicellular yeasts Multicellular moulds
Possess a cell wall. Do not contain chlorophyll

Very few pathogenic in man Principally opportunistic pathogens Eg HIV, C. albicans. Most fungal infections (mycoses) are cutaneous. Systemic mycoses are life-threatening.

Tinea pedis (Athletes Foot)

Multicellular Parasites
Endoparasites include the helminth worms (flatworms and roundworms) have microscopic stages in their life cycles and are usually classed as microbial diseases. Ectoparasites include the fleas, ticks, lice and mites eg scabies form a parasitic relationship on the outside surface of the human body may also act as vectors for microorganisms.

Prion

transmissible particles no nucleic acid composed of a modified protein -proteaseresistant isoform of normal brain protein. initiates a conformational change in the host brain protein - abnormal brain protein accumulates vacuole formation - brings about disease. These unusual neurodegenerative disorders are called transmissible spongiform encephalopathies Creutzfeldt-Jakob disease (CJD) is the most wellknown of the human TSEs. It is a rare type of dementia that affects about one in every one million people each year