Microbiology the basics

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Microbiology - the basics

• A brief review

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Ericka King, E-01tutor

• efking_at_u.washington.edu

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How to study for this class

• Before class
• At least read the syllabus
• Better skim the textbook (only 1-2 pages/bug)
• Best memorize the basics about each bug
• Loa loa is a nematode in Africa that crawls
  across the eye.

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How to study for this class

• After class
• Memorize for each bug
• Name, classification, genome type for viruses
• Diseases it causes the mechanism of disease
• Names of toxins, their targets
• How to Dx it
• How to Rx it

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Resources

• Textbook
• Good on bacteria OK on viruses lousy on
  parasites
• Board review books
• My favorite microbiology made ridiculously
  simple
• Flash cards
• Commercial bug cards
• Make your own

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Here we go
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Prokaryotes vs. Eukaryotes

• No internal, membrane-bound organelles
• Small - average .8-1.5 ?m
• Ribosomes 70S
• 50S 30S subunits
• Genome
• Haploid (always)
• Circular
• Plasmids
• No sterols in plasma membrane
• Cell wall

• Membrane-bound organelles
• Larger - RBC is 8 ?m
• Ribosomes 80S
• 60S 40S subunits
• Genome
• Diploid ( usually)
• Linear
• No extrachromosomal DNA
• Cholesterol in plasma membrane
• Chitin or no cell wall

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Functions of the cell wall

• Protects bacterium from osmotic lysis
• Provides rigidity

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Gram vs. Gram -

• Cell membrane
• Cell wall peptidoglycan
• Repeating disaccharide units with 5 amino acid
  side chains
• Last 2 D-ala - D-ala
• Last D-ala removed upon crosslinking
• Cross-linked extensively by transpeptidases
• These are targets for penicillin PBPs

• Cell membrane
• Periplasmic space
• Surface structure assembly
• ?-lactamase, other enzymes
• Cell wall
• Peptidoglycan layer is thin
• No teichoic acid
• Murein lipoprotein - binds outer membrane

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Gram vs. Gram -(contd)

• Cell wall - other components
• Teichoic acid
• Antigenic determinant
• Serology
• Lipoteichoic acid
• Adhesin
• Diffusion of LMW compounds

• Cell wall
• Outer membrane
• Phospholipid bilayer
• Porins - barrier to diffusion
• Outer leaflet contains LPS
• O specific antigen (species)
• Core polysaccharide (genus)
• Lipid A -gt endotoxin

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Exceptions

• No cell wall
• Mycoplasma
• Chlamydia
• Mycolic acid in cell wall (acid-fast)
• Mycobacteria
• Nocardia

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SPORES

• G only
• Bacillus, Clostridium
• Form in response to stress
• Resistant to uv, drying, heat
• Sporulation - 6-8 hours
• Dormancy - centuries
• Germination - 90 minutes

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Other structures outside of cell wall that you
should care about

• Polysaccharide capsule defense against
  immune system
• Slime layer glycocalyx
• Flagella
• Pili
• Adhesion to surfaces
• sex

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Question 1

• The cell envelope of gram-negative bacteria
• Has a thick peptidoglycan layer
• Has a membrane outside of the peptidoglycan layer
• Has teichoic acids attached to the peptidoglycan
  layer
• Does not contain porins
• Plays a role in spore formation

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Question 1

• The cell envelope of gram-negative bacteria
• Has a thick peptidoglycan layer
• Has a membrane outside of the peptidoglycan layer
• Has teichoic acids attached to the peptidoglycan
  layer
• Does not contain porins
• Plays a role in spore formation

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Bacterial Growth

• Requirements for
• Oxygen
• Carbon
• Ions
• Temperature, pH
• Generation time
• Growth stages

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Oxygen

• To deal with oxygen, bacteria must have some/all
  of the following enzymes
• Catalase - breaks down H2O2
• Peroxidase - ditto
• Superoxide dismutase - breaks down O2-

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Oxygen continued
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Carbon

• All medically important bacteria are
  HETEROTROPHS
• Need organic compounds for energy
• Glucose is preferred energy source

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Ions etc.

• Iron
• Potassium
• Chloride
• Magnesium
• Also require nitrogen, phosphorus, sulfur

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Temperature and pH

• Temperature
• Mesophiles like human body temperature
• pH
• Neutrophiles most like neutral pH
• May neutralize acidic environment to grow (H.
  pylori)
• Some can tolerate acid/alkaline environment

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Generation time

• Time for the bacterial population to double
• Lag phase - adaptation to new environment, gene
  activation
• Log phase - growing freely
• Stationary- balance between growth death
• Death

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Question 2

• The major reason for sensitivity of strict
  anaerobes to oxygen is
• Production of superoxide ion
• Sensitivity of their ribosomes to oxidative
  stress
• Inhibition of chemotaxis
• Interference with fermentation

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Question 2

• The major reason for sensitivity of strict
  anaerobes to oxygen is
• Production of superoxide ion
• Sensitivity of their ribosomes to oxidative
  stress
• Inhibition of chemotaxis
• Interference with fermentation

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Bacterial Genetics

• Transformation
• Conjugation
• transduction

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Transformation

• Direct uptake of free DNA
• Recipient must be competent to take DNA up,
  integrate it
• Not all bacteria can do naturally can
  electroporate in lab to force it

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Conjugation

• Bacterial sex - transfer of genetic material via
  cell-cell contact
• Major mechanism for transfer of antibiotic
  resistance
• One bacterium must have F plasmid
• Encodes enzymes, proteins needed for conjugation
• sex pilus (penis)
• oriT gene
• These are called F cells
• F cells can pass plasmid to F- recipient cell,
  making it F
• Plasmid may become integrated into bacterial
  chromosome -gt Hfr
• Now, when this cell mates with an F- cell, may
  pass whole or part of genome over
• Or, integrated plasmid may be excised at a
  different site from that of integration, and gets
  linked with a few segments of chromosomal DNA -gt
  F plasmids can transfer these genes to an F-
  cell

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Transduction

• Via bacteriophages
• Generalized
• Phage enters bacterium its DNA is expressed,
  repressing bacterial DNA
• Chunks of bacterial DNA may get packaged into new
  phages in lieu of phage DNA
• These pieces get injected into a new bacterium,
  may integrate into genome
• Specialized
• Phage enters bacterium, its genome gets
  integrated into bacterial DNA -gt prophage
• Eventually becomes active prophage DNA is
  spliced out of bacterial chromosome.
• With an error is splicing, grabs some bacterial
  DNA on either side gets packaged into new phage,
  transferred to another bacterium

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Transposons

• Mobile genetic elements - pieces of DNA with
  legs
• Can insert into donor chromosome without DNA
  homology
• Insert into DNA of phages, plasmids, bacteria

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Question 3

• Bacteriophages cannot
• Survive outside the bacterial cell
• Replicate in the periplasmic space
• Encode extracellular toxins
• Transfer transposons

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Question 3

• Bacteriophages cannot
• Survive outside the bacterial cell
• Replicate in the periplasmic space
• Encode extracellular toxins
• Transfer transposons

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Question 4

• Pili are important for DNA exchange
• Via transduction
• Only between bacteria of the same strain
• In transformation-competent bacteria
• Via plasmid transfer
• Via attachment to human cells

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Question 4

• Pili are important for DNA exchange
• Via transduction
• Only between bacteria of the same strain
• In transformation-competent bacteria
• Via plasmid transfer
• Via attachment to human cells

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Gene regulation

• Constitutive expression
• Depends only on promoter sequence
• Negatively-controlled genes
• Expression is turned OFF by a repressor bound to
  operator DNA
• Inducer -gt induces expression by inhibiting
  repressor
• Co-repressor -gt inhibits expression by activating
  an apo-repressor
• Positively-controlled genes
• Expression is turned ON by an apoinducer binding
  to DNA
• Inducer -gt activates the apoinducer, induces
  expression
• Co-repressor -gt inhibits the apoinducer, inhibits
  expression

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Lac operon

• Inducer lactose
• Removes inhibition by inhibiting LacI repressor
  protein
• Negative control
• Apoinducer CAP protein
• Activates lac promoter
• Requires cAMP
• Positive control

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Bacterial morphology and classification

• Staining
• Shape
• Oxygen requirement
• Spore forming ability

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Bacterial morphology and classification

• Staining
• Shape
• Oxygen requirement
• Spore forming ability

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Gram Stain

• Large complex of purple dye deposits in thick G
  cell wall
• Skimpy G- cell wall cant hold the purple gets
  washed away by alcohol, takes red counterstain
• Gram purple
• Gram - red

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Shape

• Cocci round
• Clusters
• Chains
• Diplococci
• Bacilli rods
• Vibrio curved
• Spiral
• Pleomorphic indistinct shape

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Antibiotics

• Exploit differences between human bacterial
  cells -gt selective toxicity
• Bacteriostatic vs. bacteriocidal
• MIC - lowest concentration that inhibits growth
• Determine by disk diffusion
• MBC - lowest concentration that kills bug
• Resistance

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Antibiotics - what you should know

• Target of each class of drugs
• Name of major drugs in each class
• How bugs get resistant to those drugs
• Toxicity?

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Target cell wall
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Target protein synthesis
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Target protein synthesiscontd
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Target nucleic acids
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Target folate metabolism
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Question 5

• Bacterial resistance to vancomycin usually
  involves
• Enzymatic degradation of the antibiotic
• Enzymatic modification of the antibiotic
• Production of new cell wall transpeptidases
• Modification of the peptidoglycan subunit
• Mutations in genes encoding porins

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Question 5

• Bacterial resistance to vancomycin usually
  involves
• Enzymatic degradation of the antibiotic
• Enzymatic modification of the antibiotic
• Production of new cell wall transpeptidases
• Modification of the peptidoglycan subunit
• Mutations in genes encoding porins

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Question 6

• The target of the antibiotic rifampin is
• The ribosome
• Peptidoglycan
• Cytoplasmic membrane
• RNA polymerase
• Folate metabolism

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Question 6

• The target of the antibiotic rifampin is
• The ribosome
• Peptidoglycan
• Cytoplasmic membrane
• RNA polymerase
• Folate metabolism

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