Plasmids
Chromosome
Plasmid
Chromosome
Plasmid + Transposon
Chromosome +
transposon
Plasmid + integron
Chromosome +
transposon +
integron
Plasmid+transposon
+intergron
Antimicrobials 4: Testing and
Selection
Dr Fiona Walsh
Role of Antibiotic Therapy
• Prevention or Cure
• Cure or control
• Benefits outweigh disadvantages
• Efficient treatment
– Test bacteria sensitivity
– Understand antibiotic in human body
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
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
Diffusion of antibiotic from a paper disc
Disc
Concentration
Gradient
After
Incubation
Disc
Concentration of
antibiotic at
periphery of zone
equals the MIC
Area of
Bacterial
growth
Zone of
Sensitivity
Minimum Inhibitory Concentration
(MIC) Determination
Breakpoint
10
8
0
1
2
6
4
2
0
4
8
(mg/L)
16
1
2
4
8
16
>16
Minimum Inhibitory Concentration (mg/L)
MIC
• Breakpoint: concentration above which the isolate is described at
resistant and below which is susceptible
e.g. S < 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 > 90% of population inhibited.
Also show if resistance is emerging i.e. 10% of population resistant.
Minimum Inhibitory Concentrations
MIC 50, MIC90 and Range
MIC series
16
14
MIC90
12
MIC50
MIC (mg/L)
10
8
Series1
6
4
2
0
1
11
21
31
41
51
Bacteria 1 to 100
61
71
81
91
Etest Determination of MIC
CI
32
24
16
12
8
6
4
3
2
1.5
1
Breakpoint Test to Determine Bacterial
Sensitivity
Inoculation of Plates
Control - No drug
Ciprofloxacin (1mg/L)
Trimethoprim (4mg/L)
Cefotaxime (2mg/L)
Amoxycillin (4mg/L)
Ceftazidime (4mg/L)
Determination of
growth at fixed
concentrations
shows resistance
Gentamicin (2mg/L)
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
Minimum Bactericidal
Concentration
MIC
64
32
16
8
4
2
1
mg/l
Subculture onto drug-free agar
MBC
16
8
4
2
1
mg/l
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.
Factors which can influence
therapeutic outcome
Bacterial
Human
Inhibitory activity
Absorption
Subinhibitory activity
Distribution
Concentration-dependent
activity
Time-dependent activity
Metabolism
Bactericidal/bacteriostatic
activity
Protein-binding
Post-antibiotic effect
Resistance
Excretion
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.
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.
Pharmacokinetics
Concentration (mg/L)
Dosing interval
64
32
Dosing interval
tmax
16
8
4
t½
2
Cmax
1
0
1
2
3
Time (hours)
4
5
6
Pharmacokinetics
Concentration (mg/L)
Area Under the Curve
30
20
10 Area under
MIC90
the curve
0
4
8
12
16
20
Time (hours)
AUC = Initial concentration / Elimination rate constant
AUIC = AUC ( drug concentration) / MIC90
AUIC Preferably  250 but usable if > 125
24
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 halflives, 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.
Toxicity
The Need to Monitor Serum Levels
Dosing interval
Concentration (mg/L)
Initial dose
64
32
16
8
4
2
1
0
5
10
15
20
Time (hours)
25
30
Antibiotic Assays
Initial dose
Concentration (mg/L)
Dosing interval
64
32
Peak
16
8
4
Trough
2
1
0
5
10
15
Time (hours)
20
25
30
Post-Antibiotic Effect (PAE)
Viable Count (cfu/ml)
10000
Antibiotic
Induced death
1000
1.6 hours to increase 1 log10
100
Removal of
Antibiotic
10
1 log10 increase
3.1 hours to increase 1 log10
1
Control
0
1
2
3
Time (hours)
PAE = 3.1 - 1.6 = 1.5 hours
Due to antibiotic effect only
4
5
6
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 (10fold) 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
Post-Antibiotic Effect
• Precise mechanism is still not understood
• Examples of PAE
Drug
Conc (mg/l)
Imipenem
0.2
Ciprofloxacin
0.5
Gentamicin
5
Penicillin
0.05
PAE (hours)
2.6
2
2
1.5
Summary
• Sensitivity testing
– Advantages
– Disadvantages
• Pharmacological action of antibiotics
– Ideal drug
– Influence of factors on performance
• Drug choice
– Cheap
– Most Effective
– Least toxic