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116/11/2016 biofilms in vitro & clinics
Antibiotics and biofilm: in vitro evidence and
new clinical applications
Prof. Françoise Van Bambeke, PharmD, PhD
Pharmacologie cellulaire et moléculaireLouvain Drug Research Institute
Université catholique de Louvain, Brussels, Belgium
<www.facm.ucl.ac.be>
16/11/2016 biofilms in vitro & clinics 2
Biofilms: what are we speaking about ?
Attachment Maturation Dispersion
16/11/2016 biofilms in vitro & clinics 3
History of biofilm development in vivoNatural history of biofilm formation in vivo during the establishment
of chronic implant-associated S.aureus osteomyelitis in mice
Nishitani et al; J Orthop Res. 2015;33:1311-9
Attachment Maturation Dispersion
16/11/2016 biofilms in vitro & clinics 4
Biofilms in human infections
aCDC 1999; bLewis et al, Nat Rev Microbiol. 2007; 5:48-56
earnosethroat
mouth & teetheyelungheart
kidneygall bladderpancreas
nervous systemskinbone***
implanted medical devices
Biofilms are associated to 65a-80b % of human infections and can colonize virtually all organs …
16/11/2016 biofilms in vitro & clinics 5
Main pathogens in biofilm-related diseases
Römling & Balsalobre; J Intern Med. 2012; 272:541-61
16/11/2016 biofilms in vitro & clinics 6
Antibiotics and biofilms in clinical practice
Treatment failure is not rare…
16/11/2016 biofilms in vitro & clinics 7
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, ..])
16/11/2016 biofilms in vitro & clinics 8
In vitro evidence : models in 96-well polystyrene plates
appropriate dyes
to evaluate biomass or bacterial load
16/11/2016 biofilms in vitro & clinics 9
Quantifying biomass and metabolic activity in biofilms
crystal violet
biofilm mass
Christensen et al, Infect. Immun. 1982; 37:318–26
16/11/2016 biofilms in vitro & clinics 10
Quantifying biomass and metabolic activity in biofilms
crystal violet
biofilm mass
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
16/11/2016 biofilms in vitro & clinics 11
Kinetics of biofilm formation
Bauer, Siala et al, Antimicrob Ag Chemother. 2013;57:2726-37
6 12 18 24 30 36 42 480
500
1000
1500
2000
2500
3000
resorufin fluorescencecrystal violet absorbance
time of incubation (h)
% o
f val
ue a
t 6 h
youngbiofilm
mature biofilm
16/11/2016 biofilms in vitro & clinics 12
Pharmacodynamic model for antibiotic activity
An example with a young biofilm of S. aureus - ATCC MSSA
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
16/11/2016 biofilms in vitro & clinics 13
Pharmacodynamic model for antibiotic activity
Young vs. mature biofilm of S. aureus - ATCC MSSA
vancomycin vs mature biofilm (24h)
0
20
40
60
80
100
120
-0.5 0.0 0.5 1.0 1.5 2.0
RFCV
log10 concentration (X MIC)
% c
ontro
l val
ue
CT
Bauer, Siala et al, Antimicrob Ag Chemother. 2013;57:2726-37
vancomycin vs. young biofilm (6h)
0
20
40
60
80
100
120
-0.5 0.0 0.5 1.0 1.5 2.0
RFCV
log10 concentration (X MIC)
% c
ontro
l val
ue
CT
Emax
C25-50-75
16/11/2016 biofilms in vitro & clinics 14
Pharmacodynamic model for antibiotic activity
Comparison of antibiotic efficacy – ATCC reference strains
Bauer, Siala et al, Antimicrob Ag Chemother. 2013;57:2726-37
LZDVAN RIF
TGCMXF
DFXOXA
FUSDAP
-100
-80
-60
-40
-20
0
E max
(% re
duct
ion)
MSSA MRSA
16/11/2016 biofilms in vitro & clinics 15
Daptomycin, a lipoglycopeptide
Van Bambeke et al; Amstrong & Cohen – Infectious diseases 2016
16/11/2016 biofilms in vitro & clinics 16
Pharmacodynamic model for antibiotic activity
Bauer, Siala et al, Antimicrob Ag Chemother. 2013;57:2726-37
Comparison of antibiotic relative potency - ATCC MSSA
LZDVAN RIF
TGCMXF
DFXOXA
FUSDAP
0.0625
0.25
1
4
16
64
256
C25
(X M
IC)
MSSA MRSA
16/11/2016 biofilms in vitro & clinics 17
Delafloxacin, a new fluoroquinolone
16/11/2016 biofilms in vitro & clinics 18
Delafloxacin, a new fluoroquinolone
16/11/2016 biofilms in vitro & clinics 19
Comparison of antibiotic activity in confocal microscopy
Bauer, Siala et al, Antimicrob Ag Chemother. 2013;57:2726-37
Live/dead staining (antibiotics at 32 X MIC) – ATCC MRSA
16/11/2016 biofilms in vitro & clinics 20
Moving to clinical isolates from pandemic lineages
Siala et al, Antimicrob. Ag. Chemother. 2014, 58: 6385–6397.
16/11/2016 biofilms in vitro & clinics 21
Comparison of 2 strains & 3 antibiotics
Siala et al, Antimicrob. Ag. Chemother. 2014, 58: 6385–6397.
viability
0
20
40
60
80
100
120
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5
DFXDAPVAN
CT
biomass
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.50
20
40
60
80
100
120
CT
0
20
40
60
80
100
120
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5CT -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.50
20
40
60
80
100
120
CT
Log10 concentration (mg/L)
Perc
enta
ge o
f con
trol
val
ue
2011S027
2003/651
Huge variability in activity
among strains…
16/11/2016 biofilms in vitro & clinics 22
Comparison of 7 strains & 5 antibiotics
Siala et al, Antimicrob. Ag. Chemother. 2014, 58: 6385–6397.
linezolid
0.01
0.1
1
10
100
25% 50% 75%
vancomycin
0.01
0.1
1
10
100
25% 50% 75%
moxifloxacin
0.01
0.1
1
10
100
25% 50% 75%
delafloxacin
0.01
0.1
1
10
100
25% 50% 75%
daptomycin
0.01
0.1
1
10
100
25% 50% 75%
S028179
104651
S027S0251083
relative potency (% reduction in resorufin fluorescence)
conc
. nee
ded
to re
ach
the
spec
ified
effe
ct (m
g/L)
Daptomycin and fluoroquinolones more potent…but again, high variability among strains
16/11/2016 biofilms in vitro & clinics 23
Importance of antibiotic concentration inside biofilms for activity
Siala et al, Antimicrob. Ag. Chemother. 2014, 58: 6385–6397.
Activity in biofilm is correlated with antibiotic penetration
16/11/2016 biofilms in vitro & clinics 24
Biofilm matrix: what is it made of ?
Rabin et al., Future Med. Chem. 2015; 7:493–512
16/11/2016 biofilms in vitro & clinics 25
Adjuvants acting on the matrix
Let’s try this amphipathic molecule …It looks like a detergent, doesn’t it ?
Siala et al, Nature Communications 2016; 7:13286
16/11/2016 biofilms in vitro & clinics 26
Adjuvants acting on the matrix
But do you recognize this
molecule ?
Siala et al, Nature Communications 2016; 7:13286
16/11/2016 biofilms in vitro & clinics 27
Adjuvants acting on the matrix
Siala et al, Nature Communications 2016; 7:13286
16/11/2016 biofilms in vitro & clinics 28
Adjuvants acting on the matrix
Siala et al, Nature Communications 2016; 7:13286
16/11/2016 biofilms in vitro & clinics 29
delafloxacin -/+ caspofungin on strain 2003/651
0
25
50
75
100
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0log10 DFX concentration (mg/l)
% c
ontro
l val
ue
2003/651
CT
Siala et al, Nature Communications 2016; 7:13286
16/11/2016 biofilms in vitro & clinics 30
delafloxacin -/+ caspofungin on strain 2003/651
-1.0 -0.5 0.0 0.5 1.0 1.5 2.00
25
50
75
100
% control values
log10 DFX concentration (mg/l)CT
0
25
50
75
100
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0log10 DFX concentration (mg/l)
% c
ontro
l val
ue
2003/651
CT
Caspofungin increases efficacy and relative potency of delafloxacinagainst a recalcitrant stain
Siala et al, Nature Communications 2016; 7:13286
16/11/2016 biofilms in vitro & clinics 31
Caspofungin-fluoroquinolone combinations
Siala et al, Nature Communications 2016; 7:13286
The combination works for two FQs
and against several strains, but to different
extents …
16/11/2016 biofilms in vitro & clinics 32
Caspofungin increases FQ potency within biofilms
The combination works for two FQs and against several strains,
but to different extents …
Siala et al, Nature Communications 2016; 7:13286
16/11/2016 biofilms in vitro & clinics 33
Caspofungin increases fluoroquinolone activity in vitro and in vivo
Cat
hete
rs in
vitr
o
Siala et al, Nature Communications 2016; 7:13286
Cat
hete
rs in
viv
o
16/11/2016 biofilms in vitro & clinics 34
Caspofungin increases fluoroquinolone activity in vitro and in vivo
Caspofungin makes fluoroquinolones active at lower concentrations
Siala et al, Nature Communications 2016; 7:13286
16/11/2016 biofilms in vitro & clinics 35
Caspofungin increases fluoroquinolone penetration
CAS fluoroquinolone
penetration in biofilms
(strain dependent)
Siala et al, Nature Communications 2016; 7:13286
16/11/2016 biofilms in vitro & clinics 36
Effect of caspofungin on PNAG in biofilm matrix
CAS poly-N-acetylglucosamine content in biofilms
Siala et al, Nature Communications 2016; 7:13286
Poly-N-acetylglucosamine
16/11/2016 biofilms in vitro & clinics 37
Effect of caspofungin on PNAG in biofilm matrix
CAS poly-N-acetylglucosamine content and polymerization in biofilms
Siala et al, Nature Communications 2016; 7:13286
Poly-N-acetylglucosamine
16/11/2016 biofilms in vitro & clinics 38
IacA and polysaccharide synthesis in S. aureus
Otto et al., Nat. Rev. Microbiol. 2009; 7:555-67
Ica A is involved in N-acetylglucosamine homopolymer synthesis
16/11/2016 biofilms in vitro & clinics 39
Caspofungin, an unexpected IcaA inhibitor !
strain icaAexpression
ATCC33591 1
2011S027 1.8 ± 0.5*
2003/1083 4.0 ± 0.6 *
2009S025 2.5 ± 0.5*
2005/104 4.2 ± 0.4*
2005/179 6.0 ± 0.9*
2009S028 4.1 ± 0.2*
2003/651 16.3 ± 0.7*
CAS inhibits IcaA and increases FQ potencyin inverse proportion
to icaA expression
Siala et al, Nature Communications 2016; 7:13286
16/11/2016 biofilms in vitro & clinics 40
The antifungal caspofungin as an inhibitor of polysaccharide synthesis
Candida albicans
Adapted from Arnold, Kucer’s 6the edition
Glucan synthase
Atkin et al, FEBS Lett. 2014;588:1869-72
16/11/2016 biofilms in vitro & clinics 41
Caspofungin, an unexpected IcaA inhibitor ?
IcaA is homologous to glucan synthases (caspofungin target in fungi)
Siala et al, Nature Communications 2016; 7:13286
16/11/2016 biofilms in vitro & clinics 42
Caspofungin as a prototype for icaA inhibitors
Siala et al, Nature Communications 2016; 7:13286
16/11/2016 biofilms in vitro & clinics 43
Antibiofilm strategies under study in the lab …
Kostakioti et al. Cold Spring Harb Perspect Med 2013;3:a010306
16/11/2016 biofilms in vitro & clinics 44
New clinical applications
1. Infections on medical devices
This is not the main
reason for contamination
…
16/11/2016 biofilms in vitro & clinics 45
Biofilms on endoscopes and cleaning procedures
https://www.educationaldimensions.com/eLearn/biofilm/photograph.php
glutaraldehyde/alcool
paracetic acid
removal of air/water noozle
manual cleaninguncleaned colonoscope
16/11/2016 biofilms in vitro & clinics 46
Importance of cleaning procedure
No biofilmbiofilm
Ren-Pei et al, Am J Infect Control 2014; 42:1203-6
GARUDA A B C D E F G H I J0
5
10
15
cleaner
Nb
of b
iofil
ms
rem
oved
of >
50%
by
clea
ner
GARUDA A B C D E F G H I J0
5
10
15
cleaner
Nb
of b
iofil
ms
not a
ffect
edby
cle
aner
16/11/2016 biofilms in vitro & clinics 47
Efficacy of biofilm-removing detergents
Siala et al, unpublished
In vitro evaluation of 12 detergent solutions against 15 biofilms from different species
High variability in capacity to act upon biofilms among detergents
16/11/2016 biofilms in vitro & clinics 48
Efficacy of biofilm-removing detergents
UNTREATED CLEANER A
GARUDA CLEANER J
Siala et al, unpublished
16/11/2016 biofilms in vitro & clinics 49
Efficacy of biofilm-removing detergents
AER: Automated Endoscope Reprocessors
Siala et al, unpublished
Ex vivo efficacy of GARUDA® for endoscope cleaning
GARUDA® removes most of remaining contamination
16/11/2016 biofilms in vitro & clinics 50
New clinical applications
2. Infections on catheters
Von Eiff, Lancet ID 2002; 2:677-685
16/11/2016 biofilms in vitro & clinics 51
New clinical applications
Infections on catheters
16/11/2016 biofilms in vitro & clinics 52
Lock therapy and catheter-related infections
Lebeaux et al. Lancet ID 2014;14:146-59.
Totally implanted venous access catheters
• Closed system but accessible to colonisation• Possibility to follow colonisation
in the chamber, in the catheter, the related infection
Antibiotic lock solution
16/11/2016 biofilms in vitro & clinics 53
Antibiotic lock therapy: current practice
Lebeaux et al, Lancet ID 2014; 14:146–159
16/11/2016 biofilms in vitro & clinics 54
Antibiotic lock therapy: current practice
Lebeaux et al, Lancet ID 2014; 14:146–159
16/11/2016 biofilms in vitro & clinics 55
Antibiotic lock therapy: current practice
Lebeaux et al, Lancet ID 2014; 14:146–159
VAN/cefazolin AmpB/CASFQ/AGVAN/AG
EtOH
50-80 % success > 50 % failure
16/11/2016 biofilms in vitro & clinics 56
Antibiotic locks: clinical efficacy
Van de Wetering et al, Cochrane Database of Systematic Reviews 2013; 11
16/11/2016 biofilms in vitro & clinics 57
Anti-coagulant + antibiotic locks: clinical efficacy
Van de Wetering et al, Cochrane Database of Systematic Reviews 2013; 11
16/11/2016 biofilms in vitro & clinics 58
Lock therapy in the lab: screening of antibiotics
Fernandez-Hidalgo et al, J Antimicrob Chemother 2010; 65: 525–530
Hogan et al, Antimicrob Agents Chemother. 2016;60:5968-75.
16/11/2016 biofilms in vitro & clinics 59
Aminoglycosides + EDTA
Hogan et al, Antimicrob Agents Chemother. 2012;56:6310-18.
cont
rol
Gen
ta+
EDTA
control Genta + EDTA
Genta + EDTA : highly synergistic combination
16/11/2016 biofilms in vitro & clinics 60
Modulation of gentamicin activity: L-Arginine
Lebeaux et al, J. Infect. Dis. 2014; 210; 1357–66.
In vitro synergismO
O
O
CH2H3N
NH3
OH
H2NH3C
CH3OH
HO
H3NO NH3
O
O
O
CH2H2N
NH2
OH
HNH3C
CH3OH
HO
H2NO NH2
L-Arginine improves Genta activity by increasing pH
16/11/2016 biofilms in vitro & clinics 61
Modulation of gentamicin activity: L-Arg
Lebeaux et al, J. Antimicrob. Chemother. 2015; 70:1704-1712
In vivo synergismL-Arg; basic
pH
L-Arg; neutral
pHneutral
pH
In vivo, some of the L-Arg effects are pH independent
16/11/2016 biofilms in vitro & clinics 62
Grafting non-biocidal anti-adhesion molec. on catheters
Surface of the TIVAP
Titanium (Ti)
or Silicon (Si)
Polymer brush
Chauhan et al. J. Infect. Dis. 2014; 210:1347–1356.
Si
Si-Ti
Coating prevents bacterial adhesion
16/11/2016 biofilms in vitro & clinics 63
Grafting non-biocidal anti-adhesion molec. on catheters
Chauhan et al. Journal of Infectious Diseases 2014; 210:1347–1356.
adherence colonization at day 5
CT
Si
Si-Ti
Coating prevents bacterial adhesionand further colonization
16/11/2016 biofilms in vitro & clinics 64
New clinical applications
3. Orthopedic infections
biofilm observed in electron microscopy on a steel component of an Ilizarov device
obtained from a patient with clinical infection (S. aureus)
Bartoszewicz et al; Orttopediia Traumatologia Rehabilitacja 2007; 9:310-8
16/11/2016 biofilms in vitro & clinics 65
Evidence for biofilm in orthopedic infectionsIncreased risk of treatment failure for biofilmogenic
S. epidermidis in Device-Related Osteomyelitis of the Lower Extremity in Human Patients
Morgensten et al, J Orthop Res. 2016 doi: 10.1002/jor.23218.
16/11/2016 biofilms in vitro & clinics 66
Evidence for biofilm in orthopedic infectionsPersistent isolates of S. aureus are higher biofilms producers ….
Trouillet-Assant et al, Cellular Microbiology 2016; 18:1405–14
Biomaterials for antibiotic delivery
Available prophylactic biomaterials vehicles:
a. Collagen (hypersensitivity, poor handling)b-c: PMMA [methylmetacrylate] beads or spacers (non degradable)d. PDLLA [poly-D,L-lactide] (acidic degradation products)e. Calcium sulfates (osteoconductive)
ter Boo et al, Biomaterials 2015, 52:113-25.
a b c d e
Images: Dr. Mario Morgenstern BGU Murnau, Germany; Dr Menendez, Jobe Orthopaedic Clinic, Los Angeles, CA
16/11/2016 biofilms in vitro & clinics 67
Biomaterials for antibiotic delivery
Variable antibiotic release from commercial cements
ter Boo et al, Biomaterials 2015, 52:113-25.16/11/2016 biofilms in vitro & clinics 68
Antibiotic-loaded bone cements: clinical experience
Iarikov et al., Clin Infect Dis 2012; 55:1474-80 16/11/2016 biofilms in vitro & clinics 69
New developments: an example
Thermoresponsive Hyaluronanhydrogel
Ter Boo et al., Acta Biomaterialia 2016; 43:185–194
Rapid release of gentamicin from the gel; low serum levels
16/11/2016 biofilms in vitro & clinics 70
New developments: an example
Ter Boo et al., Acta Biomaterialia 2016; 43:185–194
Thermoresponsive Hyaluronanhydrogel
Genta-loaded hydrogelreduces infection
16/11/2016 biofilms in vitro & clinics 71
16/11/2016 biofilms in vitro & clinics 72
Take home messages
Antibiotic activity poor against biofilms due to PK/PD issues
Combinations with adjuvants effective in animal models
Prevention easier than cure …
16/11/2016 biofilms in vitro & clinics
Acknowledgments
Patrick Van DijckWafi Siala Sona Kucharíková
73
Julia Bauer
16/11/2016 biofilms in vitro & clinics
Disclosures
74
• Research grant from Melinta therapeutics (delafloxacin)
• First entreprise program of the Region wallonne with OneLIFE(Specialized medical cleaning)
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