Page 1
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>
Page 2
16/11/2016 biofilms in vitro & clinics 2
Biofilms: what are we speaking about ?
Attachment Maturation Dispersion
Page 3
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
Page 4
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 …
Page 5
16/11/2016 biofilms in vitro & clinics 5
Main pathogens in biofilm-related diseases
Römling & Balsalobre; J Intern Med. 2012; 272:541-61
Page 6
16/11/2016 biofilms in vitro & clinics 6
Antibiotics and biofilms in clinical practice
Treatment failure is not rare…
Page 7
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, ..])
Page 8
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
Page 9
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
Page 10
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
Page 11
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
Page 12
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
Page 13
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
Page 14
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
Page 15
16/11/2016 biofilms in vitro & clinics 15
Daptomycin, a lipoglycopeptide
Van Bambeke et al; Amstrong & Cohen – Infectious diseases 2016
Page 16
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
Page 17
16/11/2016 biofilms in vitro & clinics 17
Delafloxacin, a new fluoroquinolone
Page 18
16/11/2016 biofilms in vitro & clinics 18
Delafloxacin, a new fluoroquinolone
Page 19
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
Page 20
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.
Page 21
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…
Page 22
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
Page 23
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
Page 24
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
Page 25
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
Page 26
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
Page 27
16/11/2016 biofilms in vitro & clinics 27
Adjuvants acting on the matrix
Siala et al, Nature Communications 2016; 7:13286
Page 28
16/11/2016 biofilms in vitro & clinics 28
Adjuvants acting on the matrix
Siala et al, Nature Communications 2016; 7:13286
Page 29
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
Page 30
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
Page 31
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 …
Page 32
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
Page 33
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
Page 34
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
Page 35
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
Page 36
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
Page 37
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
Page 38
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
Page 39
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
Page 40
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
Page 41
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
Page 42
16/11/2016 biofilms in vitro & clinics 42
Caspofungin as a prototype for icaA inhibitors
Siala et al, Nature Communications 2016; 7:13286
Page 43
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
Page 44
16/11/2016 biofilms in vitro & clinics 44
New clinical applications
1. Infections on medical devices
This is not the main
reason for contamination
…
Page 45
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
Page 46
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
Page 47
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
Page 48
16/11/2016 biofilms in vitro & clinics 48
Efficacy of biofilm-removing detergents
UNTREATED CLEANER A
GARUDA CLEANER J
Siala et al, unpublished
Page 49
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
Page 50
16/11/2016 biofilms in vitro & clinics 50
New clinical applications
2. Infections on catheters
Von Eiff, Lancet ID 2002; 2:677-685
Page 51
16/11/2016 biofilms in vitro & clinics 51
New clinical applications
Infections on catheters
Page 52
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
Page 53
16/11/2016 biofilms in vitro & clinics 53
Antibiotic lock therapy: current practice
Lebeaux et al, Lancet ID 2014; 14:146–159
Page 54
16/11/2016 biofilms in vitro & clinics 54
Antibiotic lock therapy: current practice
Lebeaux et al, Lancet ID 2014; 14:146–159
Page 55
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
Page 56
16/11/2016 biofilms in vitro & clinics 56
Antibiotic locks: clinical efficacy
Van de Wetering et al, Cochrane Database of Systematic Reviews 2013; 11
Page 57
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
Page 58
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.
Page 59
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
Page 60
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
Page 61
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
Page 62
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
Page 63
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
Page 64
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
Page 65
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.
Page 66
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
Page 67
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
Page 68
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
Page 69
Antibiotic-loaded bone cements: clinical experience
Iarikov et al., Clin Infect Dis 2012; 55:1474-80 16/11/2016 biofilms in vitro & clinics 69
Page 70
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
Page 71
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
Page 72
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 …
Page 73
16/11/2016 biofilms in vitro & clinics
Acknowledgments
Patrick Van DijckWafi Siala Sona Kucharíková
73
Julia Bauer
Page 74
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)