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

5. 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

6. Disc Concentration Gradient 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

7. 2 0 1 4 8 16 Minimum Inhibitory Concentration (MIC) Determination Breakpoint 10 8 6 Number 4 2 0 1 2 4 8 16 >16 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 < 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.

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. MIC 64 32 16 16 8 8 4 4 2 2 1 1 mg/l mg/l 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

16. 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.

17. 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.

18. Dosing interval Dosing interval tmax t½ Cmax Pharmacokinetics 64 32 16 Concentration (mg/L) 8 4 2 1 Time (hours)

19. Area under the curve MIC90 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 > 125

20. 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.

21. Dosing interval 64 32 16 8 4 2 1 ToxicityThe Need to Monitor Serum Levels Initial dose Concentration (mg/L) 0 5 10 15 20 25 30 Time (hours)

22. Initial dose Dosing interval 64 Peak 32 16 8 4 Trough 2 1 Antibiotic Assays Concentration (mg/L) Time (hours)

23. Antibiotic Induced death 1.6 hours to increase 1 log10 Removal of Antibiotic 1 log10 increase 3.1 hours to increase 1 log10 Control Post-Antibiotic Effect (PAE) Viable Count (cfu/ml) PAE = 3.1 - 1.6 = 1.5 hours Due to antibiotic effect only

24. 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

25. Post-Antibiotic Effect Precise mechanism is still not understood Examples of PAE

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