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>
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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 …
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Main pathogens in biofilm-related diseases
Römling & Balsalobre; J Intern Med. 2012; 272:541-61
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Antibiotics and biofilms in clinical practice
Treatment failure is not rare…
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Studying antibiotic PK/PD against biofilms
Very simple ?Very complicated ?
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Static models … for dynamic studies
pegs multiwell plates
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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
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PD parameters: planktonic vs. biofilm cultures
Macià et al, Clin Microbiol Infect. 2014 Apr 26
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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
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Static models: 96-well polystyrene plates
appropriate dyes
to evaluate biomass or bacterial load
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Quantifying biomass and metabolic activity in biofilms
crystal violet
biofilm mass
Christensen et al, Infect. Immun. 1982; 37:318–26
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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
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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
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Distinguishing between static and cidal effect
CFU counting
Hardrson et al, Nature Protocols 2010; 5:1236-54
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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
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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
)
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PK/PD studies: a few exemples
Time effects
Coo
ncen
tratio
nef
fect
s
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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
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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
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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
0.5 1.0 1.5 2.0 2.5 3.0 3.5log10 concentration (X MIC)
% c
ontr
ol v
alue
daptomycin
CT0
20
40
60
80
100
120
CVRF
0.5 1.0 1.5 2.0 2.5 3.0 3.5log10 concentration (X MIC)
% c
ontr
ol v
alue
dead
life
• more active on viability than on matrix• huge difference among drugs
ATCC33591 (MRSA)
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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
Log10 concentration (X MIC)
metabolic activity biomass
Vandevelde et al, Antimicrob Ag Chemother. 2014; 58:1348-58
maximal efficacy
with maturity
moxifloxacin
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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
Log10 concentration (X MIC)
metabolic activity biomass
Vandevelde et al, Antimicrob Ag Chemother. 2014; 58:1348-58
relative potency
with maturity
moxifloxacin
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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, ..])
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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
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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
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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
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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
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In vitro dynamic models
unidirectional flow replacement
permanent fluid stirring
constant conditions
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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
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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
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Dynamic models: bioreactors
Goeres et al, Nature Protocols 2009; 4: 783-8
Drip flow reactor :• progressive, unidirectional change in medium• low shear stress
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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
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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 ??
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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)
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Dynamic models: bioreactors
Lüdecke et al, PLoS One 2014; 9(1):e84837
(non)-constant depth biofilm fermenter:• constant conditions• low shear stress• ~ biofilms on implants
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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)
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Take home messages ….
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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
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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 ?
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
11/05/2014 antibiotic PK/PD in biofilms 40
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 ?
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 ?
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Still a lot of work ahead …
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Acknowledgments
Julia Bauer Yvan Diaz IglesiasWafi Siala Nathalie Vandevelde Eugénie Basseres
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