Exercise 9 Antimicrobials MARTZ

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Exercise 9AntimicrobialsMARTZ

• Antimicrobials
• Bacterial Transformation Worksheet

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Antimicrobials

• Historical perspective
• Mid 1800s Semmelweis
• Insisted on washing hands between patients in
  hospitals, particularly maternity ward
• Mortality rate (from peurperal fever) reduced
  from 13 to less than 1
• Mid to late 1800s Lister
• Sterilized surgical instruments
• Mortality rate (from surgery) reduced from 50 to
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Antibiotics

• First attempts to use antibiotics on people
• Bad side-effects
• Determined to be too dangerous
• Ehrlich's magic bullet (1909)
• Found dyes which could penetrate microorganisms,
  but not animal tissue
• Reasoned that chemicals could attack pathogens,
  but not hosts magic bullets
• Discovered a chemical to treat syphilis
• Salvarsan or preparation 606

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Antibiotics

• Ehrlich stressed selective toxicity as key factor
  in success
• Specific to the pathogen
• Do not harm the host
• The search for antibiotics was refined
• Penicillin was introduced in 1945

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History of Antimicrobials

• Germ Theory Microorganisms can invade other
  organisms and cause disease
• Growing acceptance of the germ theory at the end
  of the 19th century led to the search for
  antimicrobial compounds.

www.molbio.princeton.edu
Alexander Fleming, who discovered that the mold
Penicillium produced a compound that lysed
Staphylococcus cells he had streaked on a plate.
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History of Antimicrobials
www.molbio.princeton.edu
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Control of Microbial Growth

• Microbial growth is never truly controlled
• Try to discourage harmful microorganisms
• Microorganisms that cause disease, spoil food,
  corrode machinery
• Try to encourage beneficial microorganisms
• Microorganisms that ferment beer or cheese,
  produce antibiotics or insulin

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Antimicrobials

• Bactericidal
• Kills the bacteria
• Bacteristatic
• Inhibits growth of the bacteria

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Antimicrobials

• Sterilizing Agents
• Kill everything
• Autoclave (heat and pressure), radiation
• Disinfectants
• Kill most things, too strong for living tissues
• Lysol, ammonia
• Antiseptics
• Prevent growth, can be used topically
• Alcohol, iodine, hydrogen peroxide
• Chemotherapeutics
• Kill microbes or prevent growth, can be ingested
• Penicillin, sulfa drugs

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Chemotherapeutics

• Can be ingested to control microbial growth
• Selectively kill or inhibit growth of harmful
  microorganisms
• Antibiotics
• Chemicals produced by one organism that kill or
  inhibit growth of other organisms

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Antibiotics Selective toxicity and target
organisms

• Bacteria
• Specific to the pathogen
• Do not harm beneficial host microflora
• Eukaryotes (such as fungi and protozoa)
• Viruses

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Known sources of antibiotics

• Mold-type fungi
• Penicillin from Penicillium species
• Cephalasporin from Cephalosporium species
• Certain strains of Bacillus
• Bacitracin, Neosporin, Polysporin
• Many strains of Actinomycetes
• Streptomycin from Streptomycetes species
• Majority of antibiotics come from these organisms

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Antibiotics Antimicrobial Susceptibility Testing

• Potential pathogen isolated from a patient
• Determine susceptibility to antimicrobial drugs
• Determine treatment of patient
• Antimicrobial susceptibility testing (AST) is
  routine procedure in clinical laboratories
• Susceptibility to antimicrobials may change
• Microorganism acquire drug resistance

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Antibiotic Combinations

• Intended to enhance inhibition
• Antibiotic synergism two (or more) antibiotics
  work together
• Enhanced inhibition
• Antibiotic antagonism two (or more) antibiotics
  work against one another
• Decreased inhibition

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Antibiotics Antimicrobial Susceptibility Testing

• AST methods
• Minimum inhibitory concentration (MIC)
• Uses serial dilutions
• Kirby-Bauer
• Uses agar plates, lawn cells, and antibiotic disks

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Minimum inhibitory concentration (MIC)

• Uses serial dilutions
• Determines minimum concentration that inhibits
  growth
• This is that point at which bacterial growth is
  bacteriostatic

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Kirby-Bauer
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Zones of Inhibition

• Zones can be resistant, intermediate, or sensitive

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Antimicrobials ExercisePurpose

• Use Kirby-Bauer method
• Compare sensitivity of Gram-negative and
  Gram-positive organisms to antibiotics
• Gram-negative organisms
• Escherichia coli and Pseudomonas aeruginosa
• Gram-positive organism
• Staphylococcus epidermidis

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Antimicrobials ExercisePurpose

• Determine antibacterial spectrum
• Broad-spectrum inhibits a wide variety of
  Gram-positive and Gram-negative bacteria
• Narrow-spectrum inhibits a limited variety of
  bacteria

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Antimicrobials ExerciseProcedure

• Prepare plates with bacterial lawn
• Apply a variety of antibiotic disks
• Each disk is impregnated with an antibiotic
• Incubate plates
• Measure zones of inhibition

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Antimicrobials Exercise Antibiotic Disks
GM 10
P 10
K 30
TMP 5
TE 30
SXT 25
PB 300
C 30
S 10
E 15
Cl 10
G 300
Figure 9.2. Antibiotic disk placement. (Shand
2003)
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Antimicrobials ExerciseZone of Inhibition
Figure 9.1. The size of a zone of inhibition
around a high potency disk reflects the
resistance or sensitivity of the bacterium.
Bacteria may also exhibit an intermediate
reaction. (Shand 2003)
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Antimicrobials ExerciseZones of Inhibition

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Antimicrobials ExerciseZone of Inhibition

• Bactericidal
• Kills the bacteria
• Bacteristatic
• Inhibits growth of the bacteria

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Bacterial Transformation

• Examine plates for growth
• Where do you expect to see growth?
• Count all colonies and fill out worksheet on p.
  8-3

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Transformation Plates
SD6 mutant lawn
1/100
U
1/10
Transformation Plate