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

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 parametersPharmacodynamic parameters

• Postantibiotic effect (PAE)– In vitro– In vivo

• Postfungal effect (PAFE)• Postantibiotic sub-MIC effect (PA SME)

– In vitro – In vivo

• Post MIC effect (PME)

• Postantibiotic leucocyte enhancement (PALE)

• Sub-MIC effect (SME)

Pharmacodynamic parametersPharmacodynamic parameters

The postantibiotic effect in vitroThe postantibiotic effect in vitro

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

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

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

Antibiotics hours

• Penicillins 1-2

• Cephalosporins 1-2

• Carbapenems 1-2

• Quinolones 1-3

• Proteinsythesis inhibitors 3-5

PAE against Gram-positive bacteria

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

PAE against P. aeruginosaPAE against P. aeruginosa

Antibiotics hours

• Penicillins 0

• Cephalosporins 0

• Carbapenems 1-2

• Quinolones 1-2

• Aminoglycosides 2-3

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

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

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

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

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

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

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

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

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

The postantibiotic effect in vivoThe postantibiotic effect in vivo

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

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

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

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

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.

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.

Postfungal effectPostfungal 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

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

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

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

PAFE on different conditions

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

The postantibiotic sub-MIC effect in vitro

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

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

The postantibiotic sub-MIC effect in vivo

The postantibiotic sub-MIC effect in vivo

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

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

Post-MIC effect (PME)Post-MIC effect (PME)

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

The post-MIC effect of benzylpenicillin against S. pneumoniae (PcR)

1

2

3

4

5

6

7

8

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

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

Postantibiotic leucocyte enhancement

Postantibiotic leucocyte enhancement

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

Sub-MIC effectsSub-MIC effects

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

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

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

Sub-MIC effectsSub-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 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

Thank you very much for your attention

CONCLUSIONSCONCLUSIONS

• Antibiotics that have long PAE / PASME or PME could maybe be dosed with longer intervals

• BUT : what about resistant subpopulations??