Plasmids ChromosomePlasmid

Plasmid + Transposon

Plasmid + integron

Plasmid+transposon +intergron

Chromosome

Chromosome + transposon

Chromosome + transposon + integron

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

After Incubation

Zone of Sensitivity

Concentration of antibiotic at

periphery of zone equals the MIC

Disc

Area of Bacterial growth

Disc

Concentration Gradient

1 2 4 8 16 >16Minimum Inhibitory Concentration (mg/L)

0

2

4

6

8

10

Num

ber

Breakpoint

(mg/L)

0

4

1

8

2

16

Minimum Inhibitory Concentration (MIC) Determination

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 ConcentrationsMIC 50, MIC90 and Range

MIC series

0

2

4

6

8

10

12

14

16

1 11 21 31 41 51 61 71 81 91

Bacteria 1 to 100

MIC (mg/L)

Series1

MIC50

MIC90

Etest Determination of MIC

16

8

4

32

24

12

6

3

21.51

CI

Breakpoint Test to Determine Bacterial Sensitivity

Trimethoprim (4mg/L)

Inoculation of Plates

Determination of growth at fixed concentrations

shows resistanceAmoxycillin (4mg/L)

Gentamicin (2mg/L)Ciprofloxacin (1mg/L) Cefotaxime (2mg/L) Ceftazidime (4mg/L)

Control - No drug

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

16 8 4 2 1 mg/l

MBC

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 HumanInhibitory activity Absorption

Subinhibitory activity Distribution

Concentration-dependent activity

Metabolism

Time-dependent activity Excretion

Bactericidal/bacteriostatic activity

Protein-binding

Post-antibiotic effect

Resistance

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.

PharmacokineticsC

on

cen

tra

tion

(m

g/L

)

0 1 2 3 4 5 6

Time (hours)

tmax

t½

Dosing interval Dosing interval

64

8

16

32

4

2

1Cmax

PharmacokineticsArea Under the Curve

Co

nce

ntr

atio

n (

mg

/L)

0 4 8 12 16 20 24

Time (hours)

20

30

10 Area under the curve MIC90MIC90

AUIC Preferably 250 but usable if > 125AUIC Preferably 250 but usable if > 125

AUC = Initial concentration / Elimination rate constant

AUIC = AUC ( drug concentration) / MIC90

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.

ToxicityThe Need to Monitor Serum Levels

Co

nce

ntr

atio

n (

mg

/L)

0 5 10 15 20 25 30

Time (hours)

Initial dose

64

81632

421

Dosing interval

Antibiotic AssaysC

on

cen

tra

tion

(m

g/L

)

0 5 10 15 20 25 30

Time (hours)

64

8

16

32

4

2

1

Initial doseDosing interval

TroughTrough

PeakPeak

Post-Antibiotic Effect (PAE)

1

10

100

1000

10000

0 1 2 3 4 5 6

Time (hours)

Removal of AntibioticRemoval of Antibiotic

Via

ble

Co

unt

(cf

u/m

l)

Control

1.6 hours to increase 1 log10

1 log10 increase

3.1 hours to increase 1 log10

Antibiotic Induced death

PAE = 3.1 - 1.6 = 1.5 hours Due to antibiotic effect only PAE = 3.1 - 1.6 = 1.5 hours Due to antibiotic effect only

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

Post-Antibiotic Effect

• Precise mechanism is still not understood

• Examples of PAE

DrugConc (mg/l)PAE (hours)Imipenem0.22.6Ciprofloxacin0.52Gentamicin52Penicillin0.051.5

Summary

• Sensitivity testing– Advantages – Disadvantages

• Pharmacological action of antibiotics– Ideal drug – Influence of factors on performance

• Drug choice– Cheap– Most Effective– Least toxic