for the PKPD approach in antimicrobial therapy Aude Ferran Toulouse May 2023 Indicators of the effect of antibiotics on bacteria In vitro MIC Minimal Inhibitory Concentration ID: 1039068
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1. Pharmacodynamic assessment for the PK/PD approach in antimicrobial therapyAude Ferran Toulouse, May 2023
2. Indicators of the effect of antibiotics on bacteriaIn vitroMIC: Minimal Inhibitory ConcentrationMBC: Minimal Bactericidal ConcentrationBacterial killing over timeIn vivoClinical outcomes: positive/negative samples, hyperthermia, leukocytosis, survival/death,...2
3. PD assessment by MIC and MBC determinations
4. MIC = Minimum Inhibitory ConcentrationIt is the lowest concentration of antibiotic that inhibits visible growth of a bacterial strain after 18-24 hours of culture at 35-37°C.=> Characterizes the bacteriostatic effect of an antibioticMIC4
5. Initial inoculum = 5.105 to 106 Colony Forming Units per milliliter (CFU/mL)5At the time of inoculation
6. According toVincent JarlierIUD Mal InfMIC = 2 mg/L6Remark: The broth usually remains clear until around 107 -108 CFU/mL.A MIC can correspond to a concentration that not fully inhibits the growthAfter 24 h of incubation
7. 96-well plate for MIC determination by micro-dilution yellow wells correspond to clear wells without bacterial growth orange wells correspond to turbid wells with bacterial growth + are positive controls without drug, - are negative control without drug or bacteria
8. Dilution of drugs in the plate
9. Preparation of a standardized bacterial inoculum of 0.5 Mc Farland (0.5 Mc Farland =1.5 x 10^8 colony forming units (CFU/ml).Pick one colony with a sterile loop and dilute it in Broth or NaCl0.9%Then, test the Optical Density (OD) with a wavelength of 500 nm of the suspension. The equivalent of 0.5Mc Farland is an OD of 0.06 to 0.08. Dilute the suspension at 1/100th before adding it in the wells
10. Reading of the MICIdentify the lowest concentration of antibiotic that inhibits visible growth of a bacterial strain after 18 -24 hours of culture at 35-37°C.
11. Use of the MIC valueFor diagnostic labs: MIC can be compared to the breakpoints for susceptibility or resistance provided by EUCAST or CLSI For PK/PD approach: MIC can be compared to Cmax or AUC obtained in animals
12. Disk diffusion method is also used to determine whether a strain is susceptible or resistant.However, it cannot be used for PK/PDbecause results are obtained in ….? Remark on disk diffusion methodmm!
13. Regression line between MIC and diameter of diffusion453525155Exemple : Penicilline GMIC(µg / ml)Diameters (mm)MIC varying 1 to 4-fold for the same diameterRemark on disk diffusion method
14. ResistantIntermediareSusceptible453525155MIC (µg / ml)Diameters (mm)Major mistakeMIC and disk diffusion
15. Checkerboard assays: a method very similar to MIC determination used to identify the interactions between two antibiotics
16. Checkerboard assays~MIC for combinations+Antibiotic A00Antibiotic BMIC B aloneMIC B when combined to AMIC A aloneMIC A when combined to BFractional Inhibitory Combination Index = Here, FICI = 0.5 + 0.5 = 1
17. Checkerboard assays If FICI< 0.5 => synergyIf FICI > 0.5 and <= 2 =>indifferenceIf FICI > or = 4 => antagonism
18. MIC = Minimum Inhibitory Concentration: lowest concentration of antibiotic that inhibits visible growth of a bacterial strain after 18 -24 hours of culture at 35-37°C.=> Characterizes the bacteriostatic effect of an antibioticMBC = Minimal Bactericidal Concentration = lowest concentration of antibiotic leaving only 0.1% or less survivors of the initial inoculum after 18-24 hours of culture at 35-37°C.=> Characterizes the bactericidal effect of an antibioticMIC and MBC18MBC are not determined routinely because the workload is far higher than for MIC determination
19. MBCThe MBC is the lowest concentrations allowing a decrease of 3 log10 CFU/mL=> The counting of bacteria at the end is unavoidable!
20. MBCCount precisely bacteria before and after MIC determination by performing serial 10-fold dilutions of the bacterial suspensions and by plating them- Then, compare the counts before and after antibiotic exposure and determine the lowest concentration allowing a 3log10 reduction = MBC
21. Serial ten-fold dilutions of the suspensionsFrom these wells, you can spread 100µL of each dilution on one agar plate per dilution. It is very fastidious and poorly reproducible between operators! Method to count bacteria when the counts are unpredictable
22. Dilution factor10010-110-310-210-410-5= (4+3+4)/3= mean of the spots with from 1 to 15 colonies dilution factormultiplication to have results in /mL instead of /10µL*** 103* 102= 3.7.105 CFU/mLSpots of 10 µL eachBacterial colonySpots of the different dilutions from the 96-well plates on the agar and reading after incubation
23. Classification of antibiotics as bactericidal or bacteriostaticwhen MBC ~ MIC => Bactericidal antibioticwhen MBC >> MIC => Bacteriostatic antibiotic 27Important remark ! A bacteriostatic antibiotic can have a great bactericidal effect when C>> MICA bactericidal antibiotic can « only » have a bacteriostatic effect when C ~ MIC
24. MIC => Characterizes the bacteriostatic effect of an antibioticMBC => Characterizes the bactericidal effect of an antibioticMIC and MBC24MIC and MBC do not give any information on:The concentration-effect relationThe effects over time
25. PD assessment by Time-Kill Curves (TKC)(also called Time-Kill Studies (TKS))
26. Assessment of the bacterial killing as a function of time and antibiotic concentrationsTime-kill curves 26Surviving bacteriaTime
27. Methods : Time-kill curves 2700.5 MIC1 MIC2 MIC4 MIC8 MICTubes incubated at 35-37°C (depending on the strain)Prepare inoculum (incubate it one or two hours without drug)Add the drugTake care of oxygenationAgitationVolume of liquidVentilated capSerial samplings of the same tube (100 µL is enough for bacterial counts)(Removal from incubator for samplings must be short)
28. Prevention of drug carry-over on agar:Washing of the samples Centrifugation = 3000 g for 6-10 minRemoval of the supernatant (be careful of the remaining volume)Addition of the same volume of NaCl 0.9% or PBS vortexAnd/or agar with active charcoal and/or cations (Efficacy of this method depends on the physico-chemical properties of the drug)Time-kill curves 28
29. Serial ten-fold dilutions of the sampleRemark : if there are too many bacteria >107-8 CFU/mL, 10-fold dilutions in microwells will fail because bacteria remain stuck to the tips Solution: make a pre-dilution in a larger volume before the microdilutionsEg : 10 µL of the sample in 990 µl NaCl 0.9% or PBS
30. Dilution factor10010-110-310-210-410-5= (4+3+4)/3= mean of the spots with from 1 to 15 colonies dilution factormultiplication to have results in /mL instead of /10µL*** 103* 102= 3.7.105 CFU/mLSpots of 10 µL eachBacterial colonySpots of the different dilution from the 96-well plates to the agar
31. Concentration dependent(and time dependent)(Non concentration dependent)that are time-dependentSurviving bacteriaSurviving bacteriaTimeTimeClassification of antibiotics
32. Concentration-dependent antibioticstheir bactericidal rate increases with the concentration of the antibioticTime-dependent antibiotics (also called concentration-independent)as soon as they are bactericidal, the time-dependent drugs act at their maximum speed (which is slow)Classification of antibiotics32
33. Concentration-dependent antibioticsAminoglycosidesQuinolones (except on some gram-positive)Time-dependent antibiotics (also called concentration-independent)Beta-lactams (except on some gram-negative)CephalosporinsMacrolidesTetracyclinesClassification of antibiotics33
34. Allows the detection of bacterial decay/re-growth phasesTime-kill curves 34Bacterial counts (CFU/ml)latencyphase ofbactericidal activityphase ofregrowth026time (h)Conc 1Conc 224
35. Early phase: 0.5 h to 6-8 hinforms about the mode of action of the antibiotic, by the temporal dynamics of the bacterial killingLate phase: > 6-8 hbacterial regrowth degradation of the antibioticselection of resistant mutantsadaptive tolerance...Time-kill curves 35
36. Examples of time-kill curves Low inoculum051015201010108106104102Medium inoculum05101520Time (h)Time (h)High inoculum051015201010108106104102Bacteria (CFU/mL)00.5x MIC1x 2x4x8x16x 32xHere, how to quantify the influence of inoculum size? Timr (h)
37. Data from Time-kill curves Can be summarized by one value calculated to describeKilling rateOverall effect over time ...Can be fully described by a mathematical modelTime-kill curves 37
38. Quantification of bacterial killing No « official » methods, no consensus Both the speed of effect and the global/total effect can be considered (depending on the mode of action of the ATB)Calculation of the initial rate of bacterial killingCalculation of the killing effect over time (hybrid result between rate and killing activity = how many? How fast?)
39. Surviving bacteriaTimeRate of bacterial killing Rate of killing = initial slopeRate of killing can be calculated between 0-1 h, 0-3h, 0-6h,…. Expressed in log (CFU/mL)/h
40. Low inoculum051015201010108106104102Medium inoculum05101520Time (h)Time (h)High inoculum051015201010108106104102Bacteria (CFU/mL)00.5x MIC1x 2x4x8x16x 32xCalculation of the initial killing ratesTimr (h)
41. Surviving bacteriaTimeRate of bacterial killing Rate of killing = initial slope-2-1012345670.0010.010.1marbofloxacin (µg/mL)Bactericidal rate ([log CFU/mL]/h)Low inoculumHigh inoculumMedium inoculum
42. Quantification of bacterial killing The easiest way to summarize an effect over time is the calculation of the Area Under the CurveEach curve will be summarized by one number (in log(CFU/mL)xh)How to calculate AUC? AUC[0-8] with drug=ATBAUC [0-8] without drug=controllog CFU/mLTime 8 h
43. Quantification of bacterial killing log CFU/mLHow to calculate AUC? 014824Count 0Count 1Count 4Count 8Count 24Time Average log count between 0 and 1h
44. log CFU/mL014824Count 0Count 1Count 4Count 8Count 24TimeArea below LOQLOQQuantification of bacterial killing How to calculate AUC? Average log count between 0 and 1h
45. Quantification of bacterial killing No « official » methods, no consensus AUC can be calculated over 8, 24, 48h,….AUC[0-8] with drug=ATBAUC [0-8] without drug=controllog CFU/mLTime 8 hSeveral calculations can be proposed from AUC
46. Low inoculum051015201010108106104102Medium inoculum05101520Time (h)Time (h)High inoculum051015201010108106104102Bacteria (CFU/mL)00.5x MIC1x 2x4x8x16x 32xCalculation of the global effectTimr (h)
47. Quantification of bacterial killing Low inoculumHigh inoculumMedium inoculum0%20%40%60%80%100%0.0010.010.1marbofloxacin (µg/mL)Bactericidal effect (%)AUC[0-8] with drug=ATBAUC [0-8] without drug=controllog CFU/mLTime 8 h
48. Quantification of bacterial killing log CFU/mLTime 8 hEffect = decrease of average inoculum size Average inoculum for controlAverage inoculum with drug
49. Time-kill curves 49Main factors that can influence the shape of the curve (~the activity of the drug)Inoculum sizeGrowth rate (depends on conditions and nutrients)Exponential growth or latency
50. 0.0010.010.11Marbofloxacin (µg/mL)Bactericidal rate (log CFU/h) 65463210-1-2Exponential growthLatencyLatency0510152025Time (h)108104102106101000.5 MIC12 48 16 32 CFU/mLExponential growth0510152025Time (h)1081041021061010Time-kill curves Influence of the growth rate on the bactericidal rate
51. Time-kill curves 51Modelling of the dataBacterial countstimeThe evolution of bacterial counts over time is also described by equation
52. PK/PD approachModelling
53. PK/PD indices PD PK Plasma concentrationsTimeAUC CmaxT>Concsummarized bysummarized by MIC, (MBC,…)One value for the PK/PD indiceComparison to the targeted value predicting efficacy
54. PD PK Plasma concentrationsTimeConcentrations over time are described by differential equationsBacterial countstimeThe evolution of bacterial counts over time is also described by equation
55. PK/PD approachIn vitro dynamic systems
56. concentrationstimeTKCMICStatic vs dynamic systemstimeconcentrations
57. Simulation of the concentration profiles (PK ) over several daystimeconcentrationsBacterial counts over time by serial samplingsDynamic systems
58. Magnetic stirrerwastebroth + bacteria+ antibioticbroth pump37°CWater bathsamplesThermostat « Opened » systems Dynamic systems
59. Simultaneous simulation of the PK and observation of bacterial counts (PD)drugExtracapillary spaceHollow Fiber sufaceDrugin/outbacteria retained in the cartridge Dynamic systems