Microbiology midterm

Types of microorganisms

1. Acellular - viruses

2. Prokaryotes - bacteria and archaea

3. Eukaryotes - protists - algae, fungi, protozoans

Basic methods in microbiology

1. Microscopy

2. Cultivation

3. Sterility

Microorganisms in health

1. Pathogenic microbes - Harmful microorganisms that can cause diseases in their host organisms.

2. Pathobionts - Microbes that are normally harmless but can cause disease when certain conditions are met.

3. Symbionts - Mutually beneficial to their host, often providing advantages like improved nutrition or protection.

4. Microbiota - The entire community of microorganisms, including bacteria, viruses, fungi, and other microbes, residing in a specific environment, such as the human body or a particular ecosystem.

Antibiotics

1. Natural products of microbial metabolism - penicillin, tetracycline, chloramphenicol/chemically modified

2. Mostly produced by molds and a group of microbes called actinobacteria

Antibiotic resistance arises due to

1. Presence of natural antibiotics in the environment

2. Mutations and a process called horizontal gene transfer

Metagenomics

Discovering uncultivable microbes directly from the environment through next generation sequencing.

Other pharmaceuticals of microbial origin

1. Steroids

2. Prebiotics and probiotics

3. Therapeutetic enzymes

4. Bio pharmaceuticals

5. Vaccine production

Recombinant DNA technology

A method that combines genetic material from different sources to create new DNA sequences with desired traits or functions.

Uses of microbes in medical diagnosis

1. As assay organisms to determine concentrations of antibiotics, vitamins, and amino acids.

2. To determine mutagenic or carcinogenic activity.

Koch’s postulates

1. The organism must always be present in every case of the disease, but not in healthy individuals.

2. The organism must be isolated from a diseased individual and grown in pure culture.

3. The pure culture must cause the same disease when inoculated into health, susceptible individuals.

The fourth postulate was added by an American plant pathologist Erwin Frink Smith in 1905, and is stated as:

4. The same pathogen must be isolated from the experimentally infected individuals.

Viruses

1. Composed of nucleic acid and proteins.

2. Naked or enveloped with glycoproteins.

3. No organelles, no cells.

4. Intracellular parasites - rely on host enzymes to replicate.

5. Will parasite on bacteria, archaea, and eukaryotes.

Viroids

Viroids ONLY infect plants and are replicated at the expense of the host cell.

Prions

1. Infectious peptides

2. Do not replicate, instead induce a conformational change of a host protein leading to the development of disease.

3. Resemble brain proteins and cause neurodegenerative disease.

Prokaryotes

1. Single-celled organisms without proper nuclear envelope.

2. Ribosome as its only organelle - everything takes place in the membrane.

3. One circular chromosome and extrachromosomal elements (plasmids).

4. Haploid, reproduce asexually by binary fission.

5. Lack of recombination → mutations and horizontal gene transfer.

Archaea

1. Prokaryotic, with no nuclear membrane, but distinct biochemistry and RNA markers from bacteria.

2. Ancient and thrive in extreme environments - high temperatures, salinity, pH.

Fungi

— Non-photosynthetic eukaryotes

— Chitlin in cell walls

1. Yeasts - unicellular, reproduce by binary fission or budding

2. Molds - multicellular, cells known as hyphae, grow into mycelium

— Dimorphic fungi - can switch between yeast and mold lifecycle

Protozoans

— Single-celled eukaryotes

— No true photosynthesis

— Considered animals

— Some pathogenic

Methods of study

Classical methods

1. Microscopy

2. Cultivation

3. Aseptic techniques

Contemporary methods

1. Genomics

2. Metagenomics

3. Single-cell genomics

Dark-field microscopy

Type of electron microscope, excludes the unscattered beam from the image, resulting in a generally dark field around the specimen.

Phase-contract microscopy

Type of electron microscope, converts phase shifts in light passing through a transparent specimen to brightness changes in the image.

Pure culture

The study of colonies; large microbial populations which arose from a single initial cell.

Isolation of microbes in pure culture

1. Streak plate

2. Pour plate

3. Agar slants

Streak plate

For the culture to be considered pure, a single colony must be transferred three times.

Pour plate

Microbes are serially diluted until single colonies are obtained and purified using streak plate.

Agar slants

— Slanting the surface of the agar gives the bacteria a greater surface area on which to grow in a test tube.

— Slants are created in test tubes that can be capped, which minimises water loss.

— Cultures can be stored for a longer time.

Culture media nutrients necessary for growth

1. Sources of carbon, phosphorous, and nitrogen

2. Growth factors (vitamins, etc.)

3. Salts and minerals required in minuscule amounts

Culture media types (states of matter)

1. Solid media - contains agar (inedible by bacteria)

2. Liquid media - used to propagate large number of bacteria

3. Semisolid media - allows bacteria to be incorporated within the medium

Culture growth media types

1. Complex growth media - no exact composition (ex. Blood)

2. Synthetic (chemically defined) growth media - exact composition known

3. Selective growth media - promotes growth of certain species and represses the growth of others

4. Differential growth media - contains compound only certain species can eat and changes colour when digested

Most common growth media for medical use

1. Blood agar - complex, used to isolate general pathogens and bacteria that will eat anything

2. Chocolate agar - ‘cooked’ blood agar, used to release growth factors present in blood cells

3. Mueller-Hinton agar - standard growth medium for antibiotic testing

4. Saburaud-dexterose agar - standard growth medium for fungal growth

The great count plate anomaly

States that 99.9% of microbes present in the environment cannot be obtained in pure culture. Microbes that grow in culture are often not important for the community and are rare.

Cultivation independent techniques

1. Genomics - studies individual organisms (isolates)

2. Metagenomics - DNA isolated directly from the environment followed by next-generation sequencing. Allows study of individual genes (diversity) or reconstruction of complete genomes (MAGs - metagenomic assembled genomes)

3. Single-cell genomics - involves sorting individual cells microorganisms on a chip and sequencing the DNA present in the single cell.

Removal of microorganisms

1. Sterilisation - destruction of all microbes including spores

2. Disinfection - removal of pathogenic microorganisms but not spores

3. Antisepsis - a process in which bacteria is prevented from growing but not killed (ex. Iodine solution)

Methods of sterilisation

1. Dry heat

2. Moist heat

3. Filtration

4. Radiation (UV-light)

Dry heat sterilisation

1. Inoculation loops, needles - open flame

2. Laboratory glassware - hot oven (180-220C, 3h)

Moist heat sterilisation

1. Steam under pressure (121C, 20m)

2. Destroys spores

3. Autoclave - pressure cooker

4. Sterilises glassware and media

5. Denatures cellular proteins

Monitoring autoclaves

1. Physical method - measures temperature using thermometer

2. Chemical method - uses a heat sensitive chemical that changes colour at the right temperature and exposure time, autoclave tape.

3. Biological method - ensuring spore-bearing organisms are killed during sterilisation.

Filtration sterilisation

1. Sterilisation of fluids that would break down in heated - antibiotics, sera

2. Conducted by passing the liquid through a filter with pores small enough to keep the microbes on the filter

Radiation (UV-light) sterilisation

1. Used for enclosed areas - inoculation rooms

2. Used for sensitive materials

3. Damages DNA

High-level disinfectants

1. As effective as sterilisation

2. Glutaraldehyde, hydrogen peroxide, peracetic acid, chlorine compounds.

3. Disinfects surgical instruments, endoscopes

Intermediate-level disinfectants

1. May not be effective against spores

2. Alcohols, iodophors, phenolics

3. Disinfects body surfaces (a,i), medical devices (p)

4. 70% alcohol more effective than 90%

Low-level disinfectants

Quaternary ammonium compounds, ex. contact solution

Which of the following chemicals may be as effective as sterilisation?

High-level disinfectants - glutaraldehyde, hydrogen peroxide, peracetic acid, and chlorine compounds

Prokaryotic cell shapes

1. Coccus - sphere

2. Bacillus - rod

3. Spirillum - spiral

Pleomorphic - tend to change shape as they grow/combination of multiple shapes

Basic arrangements

Arise from the ability of the cell to divide across x, y, and z axes.

1. Diplo - two

2. Strepto - chain

3. Staphylo - cluster

Mechanisms that drive bacterial shape

1. Cell division and segregation

2. Attachment to surfaces

3. Passive dispersal

4. Active motility

5. The need to escape predators

The cytoplasm

1. Water (80%), nutrients, wastes, enzymes, gases, inorganic ions

2. Small (70S) ribosomes in archaea and bacteria

3. Dispersed throughout the cytoplasm

Features of plasmids

1. Extrachromosomal elements

2. Circulator or linear molecules capable of independent replication

3. Cells can increase its number of plasmids without dividing

4. Often contain genes that give advantage in a given environment.

5. Episomes - plasmids integrated into the genome

6. May be transferred among bacteria

Cell membrane

1. Selectively permeable barrier that encloses the cell

2. Lipid bilateral is fluid and elastic → fluid mosaic

3. All bacterial process occur in the membrane

Molecular structure of the cell membrane

Hydrophilic polar head connected to hydrophobic fatty acid tails through glycerol linkage. Form a bilayer.

Other membrane components

1. Lipids - increase rigidity and flexibility

   a. Sterols in eukaryotes

   b. Cyclic hopanoids in prokaryotes

2. Proteins - 60% of the membrane

Types of membrane proteins with respect to location

1. Integral proteins - span the entire membrane, often act as receptors and transfer signals. Hydrophobic amino acid regions embedded in the membrane, hydrophlic regions located outside the membrane.

2. Peripheral proteins - No hydrophobic regions, hydrophilic amino acids on the surface prevent them from being sucked into the membrane. Can detach/reattach in response to a signal.

Main membrane functions

1. Permeability barrier

2. Protein anchor

3. Energy conservation

Membrane as a permeability barrier

Strict control of transport is orchestrated by carrier proteins. Water and lipid-soluble small compounds are the only compounds that passes freely.

1. Simple diffusion

2. Facilitated diffusion

3. Primary active transport

4. Secondary active transport

Membrane as a protein anchor

1. Receptors relay signal between the cell’s exterior and interior

2. Enzymes have many activities

3. Adhesion molecules identify and interact with other cells

4. Transport proteins move molecules and ions across the membrane

Importance of carrier proteins

Uptake against the concentration gradient is necessary and mediated by carrier proteins.

The classes of membrane-transporting systems

1. Simple transporters - transport substances without chemical modification

2. Group translocation - requires phosphorylation of the transported substance

3. ABC system - couples the energy of ATP binding and hydrolysis to substance transport

Types of transport events

1. Uniport - Unidirectional transport of a molecule

2. Symport - Transport of a substance along with another substance, frequently a proton

3. Antiport - Transport of two substances in opposite directions

Membrane potential

A difference in electric potential between the interior and the exterior of a biological cell, allows formation of proton motive force.

Proton motive force consists of

1. Ion transporters - actively pushes ions across the membrane and establishes concentration gradient, acts like a battery.

2. Ion channels - allows ions to move across the membrane down the concentration gradient, acts like a resistor.

Cell wall

1. The outermost boundary of bacterial cells, very important to prokaryotes.

2. Turgor pressure (balances the osmotic pressure difference between the interior and exterior): 2 atm in gram-negative cells.

Prokaryotes without cell wall: either intracellular parasites or posses a unique membrane (lipoglycan)

Unique structure:

1. One of the few bidimesional polymers in nature

2. Just one molecule can make it a rigid container.

Cell wall functions

1. Prevents osmotic lysis

2. Maintains cellular shape

3. Provides sufficient stability, but elastic enough to allow growt

4. Enables communication with the environment

5. Enables cellular of septum during cellular division

6. Permeability barrier (in gram-negative cells)

7. Provides motility (anchors flagella)

Complex peptidoglycan structure

Polymer backbone

1. Alternating residues of sugars NAG and NAM

   NAG - N-acetyl-glucosamine

   NAM - N-acetyl-muramic acid

2. A four-aminoacid peptide (both D- and L-aa) connected to carboxyl group of NAM

3. Many bacteria substitute diaminopimelic acid at third position with another (L-Lys)

Peptidoglycan synthesis

1. Inside - Soluble substrates are activated and peptidoglycan units are built

2. Membrane - Activated units are attached and assembled on the undecaprenol phosphate membrane pivot

3. Outside - The peptidoglycan units are attached to, and cross-linked into, the peptidoglycan polysaccharide

Problems and solutions to peptidoglycan synthesis

Fairly large precursors must be delivered across the membrane. The synthesis must take part on the outside of the membrane, where there is no ATP

1. Construct energetically rich units of peptidoglycane. Park nucleotide → UDP-N-acetylmuramyl pentapeptide. (3)

2. Use the conveyer belt (4-7)

   - Undecaprenol (bactoprenol) is a membrane lipid carrier. Attach Park nucleotide to it, and it becomes a lipid.

   - Assemble the unit on the conveyor belt.

   - Flip it to the other side.

3. Insert into existing peptidogylcan (8)

   - Use transglucosylases to insert and link new monomers into the breaks in peptidoglycan.

   - Use transpeptidases to cross-link the peptidoglycan.

   - Both reactions transfer bond energy, without the need for ATP.

Gram-positive cell wall

Thick, multilayered, mainly peptidogylcan.

1. Trichroic acid - partially penetrates peptidoglycan

2. Lipoteichoic acid - lipid attached to it to anchor itself into the cytoplasmic membrane

3. Trichroic and lipoteichoic acids regulate autolytic cell-wall enzymes → maintain shape

Exoenzymes

Break down complex nutrients on the outside of gram-positive cell walls allowing them to diffuse through.

Gram-negative cell wall

1. Peptidoglycan is thin

2. Embedded in periplasm

3. Contains an outer membrane

Gram-negative outer membrane

1. Inside - phospholipids

2. Outside- lipopolysaccharide LPS, attached to the peptidoglycan by lipoprotein

Lipopolysaccharide (LPS, endotoxin)

1. Contributes to the structural integrity of the cell

2. Protects the membrane from certain chemical attacks

3. Increases the negative membrane charge

4. Plays a role in surface adhesion, phage sensitivity.

Components of lipopolysaccharide

1. Lipid A

2. Inner core

3. Outer core

4. O-antigen

Lipid A

1. Toxic component of lipopolysaccharide

2. Responsible for endotoxin activity (arouses immune system)

3. Unique to gram-negative bacteria!!

Core polysaccharide

1. Branched polysaccharide of 9 to 12 sugars.

2. Essential for structure and viability.

3. Contains an unusual sugar, 2-keto-3-deoxy-octanoate (KDO) and is phosphorylated.

O-antigen

1. Long, linear polysaccharide attached to the core.

2. Species-specific - certain e.coli are nontoxic, other strains are and differ in the sequence of the o-antigen.

Nutrient transport in gram-negative bacteria

1. No exoenzymes

2. Periplasm contains transport systems for ions, protein, and sugars

3. These are degraded by periplasmatic enzymes

4. Porins traverse entire cell wall an allow diffusion of small hydrophobic molecules.

Gram stain

1. Alcohol decolorises gram-negative cell walls because of its lacks of peptidoglycan.

2. Gram-positive cells have thick cell walls and too much dye that cannot be removed easily.

The Ziehl-Neelson/acid-fast stain

Used to stain mycobacteria (tuberculosis, leprosy), which have thick and waxy cell wall and will not stain using gram.

Alternative cell wall structures

1. Mycobacteria (tubercolosis)

   - Peptidoglycan intertwined with arabinogalatan

   - Surrounded by wax-like lipid Croat of mycolic acids

   - Only stained using acid-fast stain

2. Mycoplasma (‘walking’ pneumonia, phylum Tenericutes)

   - Have no cell wall, but incorporate host steroids into membrane to achieve rigidity

   - The membrane is three-layered

   - Evolved from gram-positive bacteria that lost their cell wall

3. Thermoplasma

Bacterial cytoskeleton proteins

1. FTsZ - homologous to tubulin - forms a ring during cell division - formation of septum

2. MreB - homologous to actin - helical - determine rod shape

3. Crescentin - homologous to lamin and keratin - bends the cell

Endospores

1. Found only in Clostridium and Bacillus*, gram-positive bacteria

2. Produced under hostile conditions from vegetative cells

3. Extremely resistant to environmental conditions

4. High concentration of calcium bound to dipicolinic acid → increased DNA stabilisation

5. Outer coat made of keratin-like proteins protects the spore.

Formation of endospore

Germination of endospore

Triggered by an environmental change which initiates gene expression, completed in 6 hours.

1. Activation

2. Germination

3. Overgrowth

Surface structure of bacteria

1. Capsule

2. Slime layer - loosely packed polysaccharide layer, easily removed from the cell

3. S-layer - composed of proteins or glycoproteins, provides rigidity, cell shape, protection from environmental changes and predators

Capsule

1. Tightly backed polysaccharide or protein layer

2. Protects the bacterium from host immune response

3. Sticky, promotes adhesion to host surfaces

4. Promotes formation of biofilm - bacteria embedded in polysaccharide are protected from antibiotics and host defence.

Structures outside of the cell wall

1. Fimbriae

   - Numerous extensions composed of protein pilin

   - Help bacteria attach to surfaces and hosts

2. Pili

   - Longer than fimbriae

   - Conjugative pili participate in DNA exchange (sex)

   - Type IV pili enable twitching motility

Bacterial flagellum

Turns clockwise or counterclockwise allowing bacteria to swim.

1. Hollow tube is made up of flagellin protein.

2. The hook allows its axis to point away from the cell.

3. A shaft runs between the hook and the basal body passing through a series of rings that embed it in the membrane and connect it with the motor.

4. The motor is powered by proton energy, H+ or Na+ flux.

Chemotaxis

1. Movement towards the attractant or away from the repellent using the flagellum

2. Orchestrated by bacterial chemoreceptors

3. Attachment of the molecule phosphorylates/methylates the receptor → activates the protein pathway.

4. Movement becomes biased towards counter clockwise rotation

Other types of locomotion in bacteria

1. Corkscrew motility - endoflagella put torsion on the entire cells

2. Gliding - does not require flagella, observed on surfaces, provided by surface proteins or slime

Biofilm formation

In a biofilm, bacterial cells are embedded in extracellular polysaccharide, making them particularly resistant to environmental conditions.

1. Pioneer cells attach to the surface through designs, fimbriae of extracellular polysaccharides.

2. Other cells are attracted to the biofilm.

3. Cells are kept at distance by polysaccharide molecules with small water channels allowing exchange of water and nutrients.

4. Protected from desiccation, antibiotics, immune system.

Bacterial classification

1. Genus name (capitalised) - species name (never capitalised)

2. Species are designated by biochemical and other phenotypic criteria and by DNA relatedness

3. Strains are category below species level and are classified below by serotyping, enzyme typing, identification of virulence factors, characterisation of plasmids, protein patters, or nucleic acids.

Identifying a microorganism

1. Colony morphology and staining

2. Metabolic examination

3. Use of bacterial viruses - bacteriophages

4. Use of serology - antibody - antigen reactions

5. Genetic differentiation - DNA hybridisation, PCR

Bacterial replication and division cycle

Occurs through binary fission - splitting into two cells.

1. Daughter cells must be capable of life

2. Have chromosomes

3. Have mRNAs, tRNAs, ribosomes, and cytochromes

Cellular division of gram-negative bacteria

1. Gram-negative cells have an outer membrane and divide by constriction, followed by membrane fusion.

2. The ring separating the cells is formed by a FtsZ protein.

Cellular division of gram-positive bacteria

1. Gram-positive cells have a thick cell wall and must develop a cross-wall in order to divide.

2. In addition to FtsZ ring, the cross wall is made out of DivIVA protein.

Timeline of E. coli cellular division

1. Replication: 45m

2. Constriction and cell wall formation: 15m

Cells can initiate several rounds of chromosomes replication and can segregate partially replicated chromosomes into new cells - can sometimes divide every 20 minutes with the right food and aeration.

Generation time

The interval between two cell divisions, the number of cells increases exponentially.

Batch culture

A closed-system microbial culture of fixed volume.

Four phases of bacterial growth curve

1. Lag phase

2. Exponential/logarithmic phase

3. Stationary phase - number of bacteria remains the same.

4. Death phase - no more nutrients present, some cells remain viable because they can cannibalise the others.

Open culture

Resembles environments of pathogens; constant supply of nutrients and removal of waste.

Physical factors that influence growth

1. Temperature

2. pH

3. Water activity/solutes

4. Availability of oxygen

5. Availability of nutrients

Physical factors that influence growth - Temperature

1. Pathogens grow at the temperature of its host.

   - human pathogens - 37C

   - avian (bird) pathogens - 42C

2. Optimal growth is often closer to maximum temperature than minimal temperature