in vitro models for the study of antibiotic pk/pd in biofilms€¦ · 11/05/2014 antibiotic pk/pd...

111/05/2014 antibiotic PK/PD in biofilms

In vitro models for the study of antibiotic PK/PD in biofilms

Françoise Van Bambeke, PharmD, PhD

Pharmacologie cellulaire et moléculaireLouvain Drug Research Institute

Université catholique de Louvain, Brussels, Belgium

<www.facm.ucl.ac.be>

11/05/2014 antibiotic PK/PD in biofilms 2

Biofilms in human infections

aCDC 1999; bLewis et al, Nat Rev Microbiol. 2007; 5:48-56

earnose throat

mouth & teeth eye lung heart

kidney gall bladder pancreas

nervous system skin bone ***

implanted medical devices

Biofilms are associated to 65a-80b % of human infections and can colonize virtually all organs …


Main pathogens in biofilm-related diseases

Römling & Balsalobre; J Intern Med. 2012; 272:541-61


Antibiotics and biofilms in clinical practice

Treatment failure is not rare…


Studying antibiotic PK/PD against biofilms

Very simple ?Very complicated ?


Static models … for dynamic studies

pegs multiwell plates


Static models: Calgary Biofilm Device

Determination of Minimal Biofilm Eradication Concentration

(MBEC)

Ceri et al, J. Clin. Microbiol. 1999; 37:1771-6; Herrmann et al, J Infect Dis. 2010;202:1585-92


PD parameters: planktonic vs. biofilm cultures

Macià et al, Clin Microbiol Infect. 2014 Apr 26


PD parameters: planktonic vs. biofilm cultures

Takei et al, J Infect Chemother (2013) 19:504–9

MIC

MBCMBEC

MIC MBC

MBEC

Ampicillin and levofloxacin vs. H. influenzae from middle ear fluid

slowly bactericidal antibiotic: MBEC >> MBC >>MIC

rapidly bactericidal antibiotic: MBEC > MBC ~ MIC


Static models: 96-well polystyrene plates

appropriate dyes

to evaluate biomass or bacterial load


Quantifying biomass and metabolic activity in biofilms

crystal violet

biofilm mass

Christensen et al, Infect. Immun. 1982; 37:318–26


Quantifying biomass and metabolic activity in biofilms

crystal violet

resazurin

resorufin

metabolic activity

fluorescein diacetate

fluorescein

Gram(+) bacteria Gram(-) bacteria

Christensen et al, Infect. Immun. 1982; 37:318–26

Tote et al, 2008; Lett. Appl. Microbiol. 46:249–254 Wanandy et al, J Microbiol Methods 2005;60:21-30

biofilm mass


CFU counting vs. RF fluorescence

relation between fluorescenceand bacterial inoculum for S. aureus

2 3 4

6

7

8

9

10

11

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

30 min. incubation

log resorufin fluorescence

log

CFU

/ml log O

D620 nm

Van den Driessche et al, J.Micr. Meth. 2014; 98:31–4Bauer, Siala et al, Antimicrob Ag Chemother. 2013;57:2726-37;

An example for S. aureus

CFU & RF signal proportional sensitivity depending on incubation time


Distinguishing between static and cidal effect

CFU counting

Hardrson et al, Nature Protocols 2010; 5:1236-54


Distinguishing between static and cidal effect

S. pneumoniae

NADH release

NADH oxidase (NOX)

NADH oxidase

NADH NAD+

Hardrson et al, Nature Protocols 2010; 5:1236-54 Yu et al, Microbiology. 2001;147(Pt 2):431-8

CFU counting


RF fluorescence vs. NADH oxidase

Vandevelde et al, ECCMID 2014; eP288

dead bacteria onlyresidual living bacteria

An example for S. pneumoniae

-7.5 -5.0 -2.5 0.0 2.5 5.00

20

40

60

80

100

120

CLRMXF

log10 concentration (X MIC)

Res

oruf

in fl

uore

scen

ce(%

con

trol

val

ue)

-7.5 -5.0 -2.5 0.0 2.5 5.00

20

40

60

80

100

120

CLRMXF

log10 concentration (X MIC)

NA

DH

abs

orba

nce

(% c

ontr

ol v

alue

)


PK/PD studies: a few exemples

Time effects

Coo

ncen

tratio

nef

fect

s


S. aureus & S. pneumoniae models

0 2 4 6 8 10 120

25

50

75

100

125

150ATCC 49619R6

Biofilm maturity (days)A

cv (5

70nm

)6 12 18 24 30 36 42 48

0

500

1000

1500

2000

2500

3000

resorufin fluorescencecrystal violet absorbance

time of incubation (h)

% o

f val

ue a

t 6 h

S. aureus S. pneumoniae

Kinetics of biofilm formation

young biofilm

mature biofilm

young biofilm

mature biofilm


Pharmacodynamic model for antibiotic activity

An example with young biofilm of S. aureus

vancomycin

CT0

20

40

60

80

100

120

RFCV

-0.5 0.0 0.5 1.0 1.5 2.0log10 concentration (X MIC)

% c

ontr

ol v

alue

Emax « efficacy »

C25-50-75 « rel. potency »

Bauer, Siala et al, Antimicrob Ag Chemother. 2013;57:2726-37


S. aureus mature biofilms: comparison of drugs

Bauer, Siala et al, Antimicrob Ag Chemother. 2013;57:2726-37

vancomycin

CT0

20

40

60

80

100

120

RFCV

0.5 1.0 1.5 2.0 2.5 3.0 3.5log10 concentration (X MIC)

% c

ontr

ol v

alue

delafloxacin

CT0

20

40

60

80

100

120

RFCV


% c

ontr

ol v

alue

daptomycin

CT0

20

40

60

80

100

120

CVRF


% c

ontr

ol v

alue

dead

life

• more active on viability than on matrix• huge difference among drugs

ATCC33591 (MRSA)


S. pneumoniae biofilms - influence of maturity

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

day 2day 11

Log10 concentration (X MIC)

% c

ontr

ol v

alue

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120


metabolic activity biomass

Vandevelde et al, Antimicrob Ag Chemother. 2014; 58:1348-58

maximal efficacy

with maturity

moxifloxacin


S. pneumoniae biofilms - influence of maturity

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

day 2day 11


% c

ontr

ol v

alue

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120


metabolic activity biomass

Vandevelde et al, Antimicrob Ag Chemother. 2014; 58:1348-58

relative potency

with maturity

moxifloxacin


PK/PD parameters in biofilms

catheter, bone, skin, cardiac valve, …

nutrients &

oxygen

pharmacokinetics

diffusibility through the matrix bioavailability within the biofilm access to bacteria efflux out of bacteria

pharmacodynamics

bacterial responsiveness (metabolic activity of bacteria)

antibiotic expression of activity (local environment [O2 , pH, ..])


Parameters affecting antibiotic activity in biofilms

2 clinical isolates of S. aureus

Siala et al, in preparation

What makes the difference ?

viability

CT0

20

40

60

80

100

120

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5

2011S027

Surv2003/651

biomass

CT0

20

40

60

80

100

120

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5

2011S027

Surv2003/651

Log10 concentration (mg/L)

Perc

enta

ge o

f con

trol

val

ue

daptomycin


PK parameter: antibiotic penetration

Siala et al, in preparation

0 2 4 6 8 10 120

5

10

15

20

25Surv 2003/651

0 2 4 6 8 10 120

5

10

15

20

252011S027

conc

entr

atio

n in

bio

film

(% a

dded

con

cent

ratio

n)

Biofilm depth (µm)

Bodipy-DAPCTC

2011S027 Surv 2003/651

more potent if better penetration

p: 0.05

more strains


PD parameter: environmental pH

pH and fluoroquinolonespH < 6

6 < pH < 9

Irwin et al, J. Appl. Microbiol. 2013; 15:382-9

less potent in acidic biofilms

Proteus mirabilis planktonic cultures vs. biofilms


PK/PD : on the way to biofilm bkpts ?

Abbanat et al, Int J Antimicrob Agents. 2014;43:32-9

Ceftobipoleand comparators

vs. S. aureus

¼ fCmax

fCmax

2 days


In vitro dynamic models

unidirectional flow replacement

permanent fluid stirring

constant conditions


Dynamic models: bioreactors

drip flow reactor ; low shear forces

Stewart et al, PLoS One 2012;7(11):e50560

CDC reactor:• constant mixing by stirring kinetic experiments with change in medium composition over time

• high shear stress


Antibiotic activity - mimicking human exposure

Parra-Ruiz et al, J Antimicrob Chemother 2012; 67:2682–5

Planktonic cultures Biofilm (CDC reactor)

S. aureus

LZDDAP

LZD +

DAP

LZDDAP

LZD +

DAP

Simulated regimens: DAP (10 mg/kg once daily) / LZD (600 mg twice daily)

CDC reactor

Combination more useful against biofilm than planktonic bacteria



Goeres et al, Nature Protocols 2009; 4: 783-8

Drip flow reactor :• progressive, unidirectional change in medium• low shear stress


Expression of antibiotic resistance

Hengzhuang et al, Antimicrob. Ag. Chemother. 2013; 57:196-204

Flow cell reactor 96-well plates, staticP. aeruginosa

Fixed conc. of ceftazidime over time

PAO1

-lactamase overproducer


Expression of antibiotic resistance

Hengzhuang et al, Antimicrob. Ag. Chemother. 2013; 57:196-204

Flow cell reactor 96-well plates, staticP. aeruginosa

Fixed conc. of ceftazidime over time

PAO1 time-dependent

-lactamase overproducer concentration-dependent ~ antibiotic access ??


Selection of resistant populations

Macià et al, Antimicrob. Ag. Chemother. 2013; 55:5230–7

P. aeruginosa

Amplification of mutator population during antibiotic treatment and accumulation of resistance mechanisms

PAO1 hypermutator

Fixed conc. of ciprofloxacin over time (MPC)



Lüdecke et al, PLoS One 2014; 9(1):e84837

(non)-constant depth biofilm fermenter:• constant conditions• low shear stress• ~ biofilms on implants


Selection of resistant populations

Hill et al, J. Antimicrob. Chemother. 2010; 65:1195-206

Constant depth fermenter

Low activity at clinically-relevant concentration

Fixed antibiotic conc. over time; multispecies biofilm (strains isolated from human wounds)


Take home messages ….


Conclusions

many methods to evaluate biomass / bacterial survival

no real consensus on the best options

many models to grow biofilms in vitro

comparison between studies difficult

more relevant model ?

Smith et al, Eur J Clin Microbiol Infect Dis 2013 ; 32:1327–32


Conclusions






antibiotic activity on biofilms <<< planktonic bacteria

no or limited effect on the matrix

determining PK parameters: diffusion / bioavailability

determining PD parameters: expression of activity / bacterial responsiveness


Conclusions






antibiotic activity on biofilms <<< planktonic bacteria

no or limited effect on the matrix

determining PK parameters: diffusion / bioavailability

determining PD parameters: expression of activity / bacterial responsiveness

combination with agents modifying matrix properties or bacterial metabolic state ?


Still a lot of work ahead …


Acknowledgments

Julia Bauer Yvan Diaz IglesiasWafi Siala Nathalie Vandevelde Eugénie Basseres

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in vitro models for the study of antibiotic pk/pd in biofilms€¦ · 11/05/2014 antibiotic pk/pd...

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