Plasmids

1
Plasmids
Chromosome
2
Antimicrobials 4 Testing and Selection

• Dr Fiona Walsh

3
Role of Antibiotic Therapy

• Prevention or Cure
• Cure or control
• Benefits outweigh disadvantages
• Efficient treatment
• Test bacteria sensitivity
• Understand antibiotic in human body

4
Objectives of lecture

• Sensitivity/Resistance testing methods
• Pharmacokinetics
• Science of time course of drug in body
• Increase effectiveness/reduce toxicity
• Pharmacodynamics
• Relationship between drug concentration at site
  of infection and pharmacological response

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Sensitivity tests

• Susceptible or resistant to antibiotic
• MIC Minimum inhibitory concentration
• MBC Minimum bactericidal concentration
• Minimum concentration required to inhibit growth
• Disc diffusion
• Agar dilution
• Etest
• Breakpoint MIC

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Diffusion of antibiotic from a paper disc
After
Incubation
Area of
Bacterial
growth
Disc
Concentration of
Zone of
antibiotic at
Sensitivity
periphery of zone
equals the MIC
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Minimum Inhibitory Concentration (MIC)
Determination
Breakpoint
10
8
6
Number
4
2
0
1
2
4
8
16
gt16
Minimum Inhibitory Concentration (mg/L)
(mg/L)
8
MIC

• Breakpoint concentration above which the isolate
  is described at resistant and below which is
  susceptible
• e.g. S lt 8mg/L R 8mg/L
• Breakpoint 8mg/L
• Range Lowest to highest MIC for population
• MIC50 Median for series of MICs
• MIC90
• MICs of population ordered from lowest to highest
• MIC value of the strains that appears 90 up the
  series.
• Antibiotic considered to be successful if gt 90
  of population inhibited.
• Also show if resistance is emerging i.e. 10 of
  population resistant.

9
Minimum Inhibitory ConcentrationsMIC 50, MIC90
and Range
MIC90
MIC50
10
Etest Determination of MIC
11
Breakpoint Test to Determine Bacterial Sensitivity
12
Evaluation of Laboratory Tests

• MIC test on plates is the best
• Time consuming and costly
• Most detailed
• Disc test/Etest is easiest
• Requires more skill to interpret
• Breakpoint
• Least skill required
• Technique must be exact
• Can be read by computer
• Large amounts of data

13
Minimum Bactericidal Concentration
Subculture onto drug-free agar
MBC
14
Pharmacokinetics/Pharmacodynamics

• General terms for any drug, not antibiotic
  specific
• The term pharmacokinetics is used to define the
  time course of drug absorption, distribution,
  metabolism and excretion.
• The term pharmacodynamics refers to the
  relationship between drug concentration at the
  site of action and pharmacologic response.
• However, when we apply these principles to
  antimicrobial therapy there are a number of
  factors that can alter the predicted outcome of
  therapy.

15
Factors which can influence therapeutic outcome
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Phamacokinetic Definitions

• Clearance is the removal of the drug from plasma
  and relates the rate at which a drug is given and
  eliminated to the resultant plasma levels
  (volume/time)
• Cmax is the maximum concentration reached at the
  site of infection, usually taken as the peak
  serum level.
• tmax is the time taken, after dosage, to reach
  the Cmax.
• Half-life (t½) is the time taken for the
  concentration of the drug in the plasma to
  decrease by half. This is often used as an
  indicator as to how often the drug should be
  administered.

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Phamacokinetic Definitions

• Area Under the Curve (AUC) is the parameter that
  links clearance to dosing. It is easily
  calculated Initial concentration / Elimination
  rate constant.
• Area Under the Inhibitory Curve (AUIC) is an
  antimicrobial adaptation of AUC, it refers to the
  concentration of the drug which is able to exert
  antibacterial activity over a given organism for
  a specific time. The AUIC is the drug
  concentration (AUC) divided by the MIC90 for a
  specific bacterial species.

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Pharmacokinetics
64
32
16
Concentration (mg/L)
8
4
2
1
Time (hours)
19
PharmacokineticsArea Under the Curve
30
20
Concentration (mg/L)
10
Time (hours)
AUC Initial concentration / Elimination rate
constant
AUIC AUC ( drug concentration) / MIC90
AUIC Preferably ? 250 but usable if gt 125
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Half-lives

• The half-life of the early antibiotics were quite
  short, perhaps only one hour or so. Therefore the
  antibiotic had to be administered many times per
  day.
• With oral versions, this causes problems with
  patient compliance and with parenteral versions,
  this becomes expensive in resources.
• Increasingly, the newer antibiotics have much
  longer half-lives, some up to 33 hours.
• This means that the patient needs to be dosed
  just once a day in order to maintain sufficient
  drug concentrations.

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ToxicityThe Need to Monitor Serum Levels
Initial dose
Concentration (mg/L)
0
5
10
15
20
25
30
Time (hours)
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Antibiotic Assays
Concentration (mg/L)
Time (hours)
23
Post-Antibiotic Effect (PAE)
Viable Count (cfu/ml)
PAE 3.1 - 1.6 1.5 hours Due to antibiotic
effect only
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Quantification of Post-Antibiotic Effect (PAE)

• The standard equation for PAE is
• PAE (hours) T - C
• T is the time required for the count of cfu to
  increase 1 log10 (10-fold) above the count
  immediately seen after drug treatment
• C is the time required for the count to
  increase 1 log10 in an untreated control culture
• PAE measures the time to reach normal logarithmic
  growth

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Post-Antibiotic Effect

• Precise mechanism is still not understood
• Examples of PAE

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Summary

• Sensitivity testing
• Advantages
• Disadvantages
• Pharmacological action of antibiotics
• Ideal drug
• Influence of factors on performance
• Drug choice
• Cheap
• Most Effective
• Least toxic