In Vitro models of Infections: the postantibiotic and sub-MIC effects in vitro and in vivo
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In Vitro models of Infections:the postantibiotic and sub-MIC
effects in vitro and in vivo
In Vitro models of Infections:the postantibiotic and sub-MIC
effects in vitro and in vivo
Inga Odenholt, MD., Ph.D.
Department of Infectious Diseases
University hospital
Malmö
Sweden
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Pharmacodynamic parametersPharmacodynamic parameters
• Postantibiotic effect (PAE)– In vitro– In vivo
• Postfungal effect (PAFE)• Postantibiotic sub-MIC effect (PA SME)
– In vitro – In vivo
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• Post MIC effect (PME)
• Postantibiotic leucocyte enhancement (PALE)
• Sub-MIC effect (SME)
Pharmacodynamic parametersPharmacodynamic parameters
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The postantibiotic effect in vitroThe postantibiotic effect in vitro
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Postantibiotic effect; PAE in vitro
Postantibiotic effect; PAE in vitro
Definition:• Suppression of bacterial growth after short
exposure of organisms to antibioticsPAE=T-CT= The time required for the exposed culture to increase one log10 above the count observed immediately after drug removalC= The corresponding time for the unexposed control
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Postantibiotic effect
3
4
5
6
7
8
9
0 2 4 6 8 10 12 h
log
10
cfu
/mL
Control
PAE
2.3 h
Odenholt et al. SJID, 1988
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Postantibiotic effectin vitro
Postantibiotic effectin vitro
The PAE is dependent on:
• Type of antibiotic
• Type of bacterial species
• Concentration of the antibiotic
• Duration of exposure
• Size of the inoculum
• Growth phase of the organism
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Antibiotics hours
• Penicillins 1-2
• Cephalosporins 1-2
• Carbapenems 1-2
• Quinolones 1-3
• Proteinsythesis inhibitors 3-5
PAE against Gram-positive bacteria
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PAE against Gram-negative bacteriaPAE against Gram-negative bacteria
Antibiotics hours• Penicillins 0• Cephalosporins 0• Carbapenems (1)• Quinolones 1-3• Proteinsythesis inhibitors 3-8• Aminoglycosides 2-4
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PAE against P. aeruginosaPAE against P. aeruginosa
Antibiotics hours
• Penicillins 0
• Cephalosporins 0
• Carbapenems 1-2
• Quinolones 1-2
• Aminoglycosides 2-3
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The PAE at different concentrations against E. coli
0
1
2
3
4
5
6
7
8
0,5 1 2 4 8 16 32
xMIC
ho
urs
Rifampicin
Tetracykline
Cefamandole
Craig & Gudmundsson, 1991
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PAE at different exposure times against S. aureus
0
1
2
3
4
5
6
0 2 4 6 8 10 12hours
PA
E (
h) Penicillin
Erythromycin
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Effect on inoculum size on the PAE
0
20
40
60
80
100
120
1 2
Min
10 9 cfu/mL
10 7 cfu/mL
10 5 cfu/mL
10 3 cfu/mL
Ciprofloxacin Tobramycin
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PAE in vitro Methods
PAE in vitro Methods
1. Viable counts
Methodological pitfalls
• may overestimate killing
• negative PAEs are common with ß-lactams and gram-negatives due to forming of filaments
• similar inocula of the control and the pre- exposed culture are desirable
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Postantibiotic effect
3
4
5
6
7
8
9
0 2 4 6 8 10 12 h
log
10
cfu
/mL
Control
PAE
2.3 h
Odenholt et al. SJID, 1988
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PAE in vitro Methods
PAE in vitro Methods
2. Optical density
Methodological pitfalls• killing cannot be measured due to a detection limit
of 106 cfu/ml
• control curves at different inocula and viable counts after drug removal are necessary to be performed to ensure that PAE culture and control are at the same inoculum
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PAE in vitro Methods
PAE in vitro Methods
3. ATP measurement
Methodological pitfalls
• bactericidal activity is underestimated due to dead but intact (not lysed) bacteria still containing intracellular ATP
• PAE is overestimated due to falsely elevated ATP content
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PAE measured with ATP
-11
-10
-9
-8
-7
0 1 2 3 4 5 6 7 8 9 h
log
10
M b
ac
teri
al
AT
P
Control
PAEDilution
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PAE in vitro Methods
PAE in vitro Methods
4. Morphology
• Phase contrast microscopy– the time it takes for the bacteria to revert to 90%
bacilli
5. 3H-thymidine incorporation
• Ultrastructural changes - the changes in structure correlates well with the PAE measured with viable counting
-correlates well with the PAE measured with viable counting
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The postantibiotic effect in vivoThe postantibiotic effect in vivo
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Postantibiotic effect in vivoPostantibiotic effect in vivo
DefinitionPAE= T-C
• T= the time required for the counts of cfu in thighs of treated mice to increase one log10 above the count closest to but not less than the time M
• C= the time required for the counts of cfu in thighs of untreated mice to increase one log10 above the count at time zero
• M= the time serum concentration exceeds the MIC
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PAE in vivoPAE in vivo• Observed in several animal models
• In vitro data are predictive of in vivo results except that in vivo PAE are usually longer due to the effect of sub-MICs and/or the effect of neutrophils
• The major unexplained discordant results are for ß-lactams and streptococci
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PAE in vivoAnimal modelsAnimal modelsPAE in vivo
Animal modelsAnimal models•Thigh infections in mice
•Pneumonia model in mice
•Infected treads in mice
•Infected tissue cages in rabbits
•Meningitis model in rabbits
•Endocarditis model in rats
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Mechanisms of PAEMechanisms of PAE
-lactam antibiotics.At least for S. pyogenes and penicillin it has been shown that PAE stands for the time it takes for the bacteria to resynthesize new PBPs
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Mechanisms of PAEMechanisms of PAE
• Erythromycin and clarithromycin:
50S ribosomal subunits were reduced during 90 min and protein synthesis during 4 h (PAE) due to prolonged binding of the antibiotics to 50S.
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Mechanisms of PAEMechanisms of PAE
• Aminoglycosides: Binding of sublethal amounts of drug enough to disrupt DNA, RNA and protein synthesis. The time it takes to resynthesize these proteins.
With a half-life of >2.5 h, the PAE disappears, reflecting a sufficient time for the repair mechanism to be restored.
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Postfungal effectPostfungal effect
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PAFE assay • Removal of the drug: 3 washes with
saline solution and centrifugation for 10 minutes after each wash.
• Colony count determination: CFU of the exposed and control within same range.
• Incubation in a spectrophotometer reader at 37 C for 48 h.
• Growth: automatically monitored: OD changes at 10 minutes intervals.
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Data analysis
Three points in the growth curve of the controls and the exposure were analyzed:
OD0: the time-point of the first
significant increase in OD.
OD20: the time-point where the
OD reached 20% of the
maximum of growth curve. OD50: the time-point where the
OD reached 50% of the
maximum of growth curve.
0.0 12 24 36 48 0
20
100
Control50
OD20
OD50
OD0
0.0 12 24 36 48 0
20
100
Control50
OD20
OD50
OD0
0.0 12 24 36 48 0
20
100
Control50
OD20
OD50
OD0
0.0 12 24 36 48 0
20
100
Control50
OD20
OD50
OD0
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Results Mean and 95% confidencial interval of the ODx for the exposed and the corresponding controls of each species at each point in the growth curve are calculated.
Presence of PAFE: Lower limit of the 95% CI of ODx of exposure > the upper limit of the 95% CI of the ODx of the corresponding control for each strain.
0
20
40
60
80
100
8 12 24 48
Time in h
% g
row
th
&*
#
0
20
40
60
80
100
8 12 24 48
Time in h
% g
row
th
&*
#
0
20
40
60
80
100
8 12 24 48
Time in h
% g
row
th
&*
#
0
20
40
60
80
100
8 12 24 48
Time in h
% g
row
th
&*
#
0
20
40
60
80
100
8 12 24 48
Time in h
% g
row
th
&*
#
0
20
40
60
80
100
8 12 24 48
Time in h
% g
row
th
&*
#
0
20
40
60
80
100
8 12 24 48
Time in h
% g
row
th
&*
#
0
20
40
60
80
100
8 12 24 48
Time in h
% g
row
th
&*
# PAFE
OD0
OD20
Exposed
Control
OD50
PAFE=T-C (t)T: time of the exposedC: time of the control
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PAFE of Amphotericin B
A. fumigatus A. ustus A. terreus A. nidulans OD0 9.94 (3/3) 3.94 (2/3) 2.53 (2/3) N.P. (0/3) OD20 8.86 (3/3) N.P. (0/3) N.P.(0/3) OD50 5.32 (3/3) 3.62 (1/3) 2.03 (2/3) N.P. (0/3)
Significant PAFE: Lower 95% CI (exposed) >Upper 95% CI (control).
N.P.: No PAFE
A. fumigatus A. ustus A. terreus A. nidulans OD0 4.05 (3/3) 1.00 (2/3) 0.64 (1/3) 1.67 (1/3) OD20 4.84 (2/3) N.P. (0/3) 0.84 (1/3) N.P. (0/3) OD50 2.95 (1/3) N.P. (0/3) N.P. (0/3) N.P. (0/3)
PAFE after 4h incubation with the drug at a concentration of 4 x MIC (Number of strains with presence of PAFE)
2.23 (2/3)
PAFE after 2h incubation with the drug at a concentration of 4 x MIC
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PAFE on different conditions
PAFE: concentration and time dependent
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PAFE for Itraconazole
Incubation period: 4, 2 and 1h
Drug concentrations: 50, 20, 10, 4, 1 and 0.25 times the MIC
No PAFE was observed for all the strains
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I. Conclusion
• The method developed seems to be useful to
measure PAFE in moulds
• OD0 was superior to OD20 or OD50:
– Least variation, reproducible
– Shortest incubation period: economic
– Maximum growth measurements not required
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II. Conclusion
For AMB:
• PAFE was dose and exposure time dependent
• No PAFE was observed after 1 h exposure at any
concentration of AMB
• No PAFE was observed at 0.25 x MIC for AMB
• A. fumigatus displayed the longest PAFE
For ITZ:
• No PAFE was present at any concentration and
exposure period
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The postantibiotic sub-MIC effect in vitro
The postantibiotic sub-MIC effect in vitro
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Postantibiotic sub-MIC effect; PA SME
Postantibiotic sub-MIC effect; PA SME
Definition• The effect of subinhibitory antibiotic concentrations on
bacteria previously exposed to suprainhibitory concentrations
PA SME= TPA-C• TPA=the time it takes for the cultures previously exposed to
antibiotics and thereafter to sub-MICs to increase by one log10 above the counts observed immediately after washing.
• C=corresponding time for the unexposed control
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PA SME of telithromycin against H. influenzae
1
2
3
4
5
6
7
8
9
10
0 3 6 9 12 15 18 21 24 h
log
10
cfu
/mL
PAE
0.1xMIC
0.2xMIC
0.3xMIC
Control
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The postantibiotic sub-MIC effect in vivo
The postantibiotic sub-MIC effect in vivo
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PAE ( PASME) in vivo of amikacin against K. pneumoniae in a thigh-infection model in
mice
PAE ( PASME) in vivo of amikacin against K. pneumoniae in a thigh-infection model in
mice
PAE
• Normal mice (half-life 19 min) 5.5 h
• Uremic mice (half-life 98 min) 14.6 h
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The PAE and PA SME of piperacillin against S. aureus in vivo
4,00
4,50
5,00
5,50
6,00
6,50
7,00
7,50
8,00
8,50
9,00
-2 0 2 4 6 8 10h
log
10
cfu
/mL
Control
PAE
PA SME
Penicillinase
T>MIC
Oshida et al. JAC, 1990
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Post-MIC effect (PME)Post-MIC effect (PME)
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Post-MIC effect; PMEPost-MIC effect; PME
Definition• The effect of sub-MICs on bacteria previously exposed
to a constant decreasing antibiotic concentration
PME=Tpme-C• Tpme= The time for the counts in cfu of the exposed
culture to increase one log10 above the count observed at the MIC level
• C= the time for an unexposed control to increase one log10
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The post-MIC effect of benzylpenicillin against S. pneumoniae (PcR)
1
2
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9
10
0 2 4 6 8 10 12 14 16 18 20 22 24 h
log
10
cfu
/mL
10mg/l
100 mg/l
Control
MIC
MIC
PME at 10 mg/l 12.9-2.3= 10.6
PME at 100 mg/l 7.5-2.3= 5.2
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Mechanism of PA SME?Mechanism of PA SME?
• The PAE of ß-lactam antibiotics seems to represent the time necessary to synthesize new PBPs. When bacteria in the PA-phase are exposed to sub-MICs, most PBPs are still inactivated and only a small amount of the drug is needed to prolong the inhibition of cell multiplication until a critical number of free PBPs are once more available
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Postantibiotic leucocyte enhancement
Postantibiotic leucocyte enhancement
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Postantibiotic leucocyte enhancement; PALE
Postantibiotic leucocyte enhancement; PALE
• Bacteria pretreated with antibiotics for a brief period of time show increased susceptibility to intracellular killing and phagocytosis
• In general, antibiotics that produce the longest PAEs exhibit maximal PALEs
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Sub-MIC effectsSub-MIC effects
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Sub-MIC effects; SMESub-MIC effects; SME
Definition
• The effect of subinhibitory antibiotic concentrations on bacteria not previously exposed to suprainhibitory concentrations
SME= Ts-C
•Ts=the time it takes for the cultures exposed to
sub-MICs to increase by one log10 above the counts
observed immediately after washing•C=corresponding time for the unexposed control
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The SME of P&G kinolon against S. pneumoniae
2
3
4
5
6
7
8
9
0 3 6 9 12 15 18 21 24h
log
10 c
fu/m
L
Control
0.1xMIC
0.2xMIC
0.3xMIC
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Sub-MIC effectsSub-MIC effects
• The minimum antibiotic concentrations that produces a structural change in bacteria seen by light or electron microscopy
• The minimum antibiotic concentration that produces a one log10 decrease in the bacterial population compared to the control
• Loss or change of bacterial toxins
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Sub-MIC effectsSub-MIC effects
• Loss of surface antigens resulting in decreased adhesion
• Increased rates of phagocytic ingestion and killing
• Increased chemotaxsis and opsonization
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Mechanism of sub-MIC effectsMechanism of sub-MIC effects
• SME probably tests the distribution of antibiotic susceptibility in the bacterial population, in which there are subpopulations that are inhibited by concentrations less than the MIC. The SME would therefore represent the time it takes for the population with the higher MIC to become dominant
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Thank you very much for your attention
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CONCLUSIONSCONCLUSIONS
• Antibiotics that have long PAE / PASME or PME could maybe be dosed with longer intervals
• BUT : what about resistant subpopulations??