Pharmacodynamic comparison of antibiotic activity against ......26/11/2017 30th ICC -Oral session OS 7 1 1 Pharmacodynamic comparison of antibiotic activity against Pseudomonas aeruginosa

126/11/2017 30th ICC - Oral session OS 7-1

Pharmacodynamic comparison of antibiotic activity against Pseudomonas aeruginosa

in models of persistent infection (intracellular infection, biofilm)

Shaunak Khandekar, Paul M. Tulkens*, and Françoise Van BambekePharmacologie cellulaire et moléculaire

Louvain Drug Research InstituteUniversité catholique de Louvain, Brussels, Belgium

www.facm.ucl.ac.be

Oral session 0S 7-1

26/11/2017 30th ICC - Oral session OS 7-1 2

Infectious diseases: No ESKAPE !

E. faecium

S. aureus

K. pneumoniae A. baumannii

P. aeruginosa

E. aerogenes


Beside resistance … persisent forms of infections

→ Protection against host defenses and antibiotics

Intracellular survival

Buyck & Van Bambeke

Biofilms

http://eyemicrobiology.upmc.com


Aim of the study

to study antibiotic activity against the reference strain PAO1 in in-vitro models of

• infected THP-1 monocytes • mature biofilms ciprofloxacin

meropenemamikacin

OpsonizationIncubation

with 10 % human serumin culture medium

Extracellular washElimination of non phagocytosed bacteria

by incubation with 50-100 X MIC gentamicin; elimination of gentamicin by washing

G G

G

G

GG

G

G

G

G

G G

Gentamicin(G)

Intracellular infectionIncubation over time

in control conditions or with antibiotics

AB

AB

AB

ABAB

AB

Antibiotic(AB)

PhagocytosisIncubation of bacteria with cells at an appropriate MOI

Determination of residual intracellularinoculum

Determination of cfu and protein content in cell lysates

cfu proteins

In vitro model of intracellular infection


Buyck et al (2016) ISBN 978-1-4939-2854-5


Setting up the model of intracellular infection

Blue: nucleusRed: Actin

Green: Bacteria

4µM

Buyck et al, AAC 2013; 57:2310-18

5 10 20 501

10

1001h 2h

0

10

20

30

40

50

60

70

bacteria per cell ratio

inte

rnal

ized

bac

teria

(105

CFU

/mg

de p

rot)

% cell death

1h2h


Activity of antibiotics against intracellular P. aeruginosa

-3 -2 -1 0 1 2 3-6

-4

-2

0

2

4 CIP

log10 extracellular conc. (x MIC)

∆lo

g C

FUfr

om in

ital i

nocu

lum • Estimation of the concentration needed to reach a specified effect

• Measure of the « intracellular MIC »

Cs (static concentration): relative potency

Cs

Emax (maximal efficacy)

• Estimation of the maximal reduction in inoculumfor an infinitely large concentration

• Measure of the killing capacity

Emax


Activity of antibiotics against intracellular P. aeruginosa

-3 -2 -1 0 1 2 3-6

-4

-2

0

2

4 CIP

extraintra

-3 -2 -1 0 1 2 3-6

-4

-2

0

2

4 MEM

extra

intra

-3 -2 -1 0 1 2 3-6

-4

-2

0

2

4 AMK

extraintra

log10 extracellular concentration (x MIC)

∆lo

g C

FUfr

om in

ital i

nocu

lum

• Emax (efficacy): intracellular <<< extracellular• Cs (relative potency): intracellular ~ extracellular

Planktonic culture

Determination of residual biofilm viability / biomass

In vitro model of biofilm


Biofilm growth4 days incubation in MHB with daily renewal of the

medium

Incubation with antibioticswide range of concentrations

during 24 h

Antibiotic(AB)

Crystal violet absorbanceMetabolisation of fluorescein diacetate


Setting up the model of biofilm

Diaz Iglesias et al, 2016 ESCMID Conference on biofilms

0 1 2 3 4 5 6 70

10000

20000

30000

40000

50000

60000

70000

0

1

2

3

4

Time (days)

Bac

teria

l via

bili t

y(fl

uore

scei

n flu

ores

cenc

e)B

iofilm biom

ass(C

V abs orb anc e)mature biofilm


Activity of antibiotics against biofilms of P. aeruginosa

• Estimation of the concentration needed to reach a specified effect

• Measure of the relative potency

C50 (conc. reducing viability of 50%): relative potency

C50

Emax (maximal efficacy)

• Estimation of the maximal reduction in viabilityfor an infinitely large concentration

• Measure of the killing capacity

Emax

-3 -2 -1 0 1 20

25

50

75

100

125

150

175CIP

log antibiotic conc. (x MIC)

% c

ontr

ol v

alue



-3 -2 -1 0 1 20

25

50

75

100

125

150

175CIP

-3 -2 -1 0 1 20

25

50

75

100

125

150

175MEM

-3 -2 -1 0 1 20

25

50

75

100

125

150

175AMK

log antibiotic conc. (x MIC)

% c

ontr

ol v

alue

• Emax (efficacy): biofilm <<< broth (no complete eradication)• C50 (relative potency): biofilm <<< broth (low effect at the MIC)



control

CIP

MEM

AMK

GFP-expressing P. aeruginosa

Antibiotics at 50 x MIC

confirmation of incomplete eradication of living bacteria


Comparison of PD parameters in these models

control

CIPMEM

AMK-5

-4

-3

-2

-1

0

-100

-80

-60

-40

-20

0

antibiotic

∆ L

og10

CFU

from

initi

al in

ocul

um(e

xtra

-intr

a)

% reduction

from control value(biofilm

)

extra

intrabiofilm

extra

intrabiofilm

extra

intrabiofilm

CIPMEM

AMK-5

-4

-3

-2

-1

0

-100

-80

-60

-40

-20

0

antibiotic∆

Log

10 C

FUfr

om in

itial

inoc

ulum

(ext

ra-in

tra)

% reduction

from control value(biofilm

)

extra

intrabiofilm

extra

intrabiofilm

extra

intrabiofilm

effect at 4 x MIC Emax

• Lower activity at equipotent concentration

• Lower Emax

intracellularly and in biofilm vs. extracellularly


Conclusions

ATIBIOTICBACTERIUM

In biofilms• Antibiotics less potent than in planktonic cultures

→ low bioavailability in biofilms• Antibiotics much less efficacious than extracellularly

→ bacteria less responsive to antibiotics

Intracellularly• Antibiotics equipotent as extracellularly

→ intracellular bioavailability similar for all classes• Antibiotics much less efficacious than extracellularly

→ intracellular bacterial less responsive to antibiotics

Innovative strategies needed to act upon these persistent forms of infections !

Documents

Pharmacodynamic comparison of antibiotic activity against ......26/11/2017 30th ICC -Oral session OS 7 1 1 Pharmacodynamic comparison of antibiotic activity against Pseudomonas aeruginosa