Transcript ICAAC WK sept 02inga..

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
Pharmacodynamic parameters
• Postantibiotic effect (PAE)
– In vitro
– In vivo
• Postfungal effect (PAFE)
• Postantibiotic sub-MIC effect (PA SME)
– In vitro
– In vivo
Pharmacodynamic parameters
• Post MIC effect (PME)
• Postantibiotic leucocyte enhancement
(PALE)
• Sub-MIC effect (SME)
The postantibiotic effect in vitro
Postantibiotic effect;
PAE in vitro
Definition:
• Suppression of bacterial growth after short
exposure of organisms to antibiotics
PAE=T-C
T= The time required for the exposed culture to
increase one log10 above the count observed
immediately after drug removal
C= The corresponding time for the unexposed
control
Postantibiotic effect
9
8
7
log10 cfu/mL
2.3 h
Control
6
PAE
5
4
3
0
2
4
6
8
10
12
h
Odenholt et al. SJID, 1988
Postantibiotic effect
in 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
PAE against Gram-positive bacteria
Antibiotics
• Penicillins
• Cephalosporins
• Carbapenems
• Quinolones
• Proteinsythesis inhibitors
hours
1-2
1-2
1-2
1-3
3-5
PAE against Gram-negative bacteria
•
•
•
•
•
•
Antibiotics
Penicillins
Cephalosporins
Carbapenems
Quinolones
Proteinsythesis inhibitors
Aminoglycosides
hours
0
0
(1)
1-3
3-8
2-4
PAE against P. aeruginosa
•
•
•
•
•
Antibiotics
Penicillins
Cephalosporins
Carbapenems
Quinolones
Aminoglycosides
hours
0
0
1-2
1-2
2-3
The PAE at different concentrations against E. coli
8
7
6
hours
5
Rifampicin
Tetracykline
Cefamandole
4
3
2
1
0
0,5
1
2
4
xMIC
8
16
32
Craig & Gudmundsson, 1991
PAE at different exposure times against S. aureus
6
5
PAE (h)
4
Penicillin
Erythromycin
3
2
1
0
0
2
4
6
8
10
12hours
Effect on inoculum size on the PAE
120
100
Min
80
10 9 cfu/mL
10 7 cfu/mL
10 5 cfu/mL
10 3 cfu/mL
60
40
20
0
1
Ciprofloxacin
2
Tobramycin
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 preexposed culture are desirable
Postantibiotic effect
9
8
7
log10 cfu/mL
2.3 h
Control
6
PAE
5
4
3
0
2
4
6
8
10
12
h
Odenholt et al. SJID, 1988
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
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
PAE measured with ATP
-7
log10 M bacterial ATP
-8
-9
Control
PAE
Dilution
-10
-11
0
1
2
3
4
5
6
7
8
9 h
PAE in vitro
Methods
4.
Morphology
• Phase contrast microscopy
– the time it takes for the bacteria to revert to 90%
bacilli
• Ultrastructural changes
-
the changes in structure correlates well with
the PAE measured with viable counting
3
5. H-thymidine incorporation
-correlates well with the PAE measured with
viable counting
The postantibiotic effect in vivo
Postantibiotic effect in vivo
Definition
PAE= 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
PAE 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
PAE in vivo
Animal 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
Mechanisms 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
Mechanisms 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.
Mechanisms 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.
Postfungal effect
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.
Data analysis
Three points in the growth curve of the controls and the
exposure were analyzed:
OD0: the time-point of the first
100
significant increase in OD.
OD20: the time-point where the
50
Control
OD50
OD reached 20% of the maximum
of growth curve.
20
OD50: the time-point where the
OD reached 50% of
the
maximum of growth curve.
OD20
OD0
0
0.0
12
24
36
48
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.
100
PAFE=T-C (t)
Control
T: time of the exposed
C: time of the control
60
Exposed
OD50
#
40
20
OD0
PAFE
OD20
&
*
48
24
12
0
8
% growth
80
Time in h
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.
PAFE of Amphotericin B
PAFE after 4h incubation with the drug at a concentration of 4 x
MIC (Number of strains with presence of PAFE)
OD0
OD20
OD50
A. fumigatus A. ustus A. terreus A. nidulans
9.94 (3/3)
3.94 (2/3) 2.53 (2/3) N.P. (0/3)
8.86 (3/3)
N.P. (0/3) 2.23 (2/3) N.P.(0/3)
5.32 (3/3)
3.62 (1/3) 2.03 (2/3) N.P. (0/3)
PAFE after 2h incubation with the drug at a concentration of 4 x
MIC
OD0
OD20
OD50
A. fumigatus A. ustus A. terreus A. nidulans
4.05 (3/3) 1.00 (2/3) 0.64 (1/3) 1.67 (1/3)
4.84 (2/3) N.P. (0/3) 0.84 (1/3) N.P. (0/3)
2.95 (1/3) N.P. (0/3) N.P. (0/3) N.P. (0/3)
Significant PAFE: Lower 95% CI (exposed) >Upper 95% CI (control).
N.P.: No PAFE
PAFE on different
conditions
10
PAFE (h)
8
6
4
4x
1x
0
4h
2h
MI
C
2
1h
Exposure tim e
PAFE: concentration and time dependent
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
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
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
The postantibiotic sub-MIC
effect in vitro
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
PA SME of telithromycin against H. influenzae
10
9
8
log10 cfu/mL
7
6
5
PAE
0.1xMIC
4
0.2xMIC
0.3xMIC
Control
3
2
1
0
3
6
9
12
15
18
21
24
h
The postantibiotic sub-MIC
effect in vivo
PAE ( PASME) in vivo of amikacin against K.
pneumoniae in a thigh-infection model in
mice
• Normal mice (half-life 19 min)
PAE
5.5 h
• Uremic mice (half-life 98 min)
14.6 h
The PAE and PA SME of piperacillin against S. aureus in vivo
9,00
8,50
8,00
log10 cfu/mL
7,50
7,00
Control
PAE
PA SME
6,50
6,00
5,50
Penicillinase
5,00
4,50
T>MIC
4,00
h
-2
0
2
4
6
8
10
Oshida et al. JAC, 1990
Post-MIC effect (PME)
Post-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
The post-MIC effect of benzylpenicillin against S. pneumoniae (PcR)
10
9
8
log10 cfu/mL
7
MIC
10mg/l
100 mg/l
Control
6
5
4
PME at 10 mg/l 12.9-2.3= 10.6
3
PME at 100 mg/l 7.5-2.3= 5.2
2
1
0
2
4 MIC
6
8
10
12
14
16
18
20
22
24 h
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
Postantibiotic leucocyte
enhancement
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
Sub-MIC effects
Sub-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
The SME of P&G kinolon against S. pneumoniae
9
8
log10 cfu/mL
7
6
Control
0.1xMIC
0.2xMIC
0.3xMIC
5
4
3
2
h
0
3
6
9
12
15
18
21
24
Sub-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
Sub-MIC effects
• Loss of surface antigens resulting in
decreased adhesion
• Increased rates of phagocytic ingestion
and killing
• Increased chemotaxsis and opsonization
Mechanism 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
Thank you very much for your
attention
CONCLUSIONS
• Antibiotics that have long PAE / PASME or
PME could maybe be dosed with longer
intervals
• BUT : what about resistant subpopulations??