C. albicans Biofilm Resistance - Fraunhofer. albicans Biofilm Resistance A Multiple Mechanism...
Transcript of C. albicans Biofilm Resistance - Fraunhofer. albicans Biofilm Resistance A Multiple Mechanism...
Biofilms
• 3 dimensional microbial community = network of cells, adherent to each other and another surface and encapsulated within an extracellular matrix
• Phenotypically distinct – Growth rate = slower – Adherent – Matrix – Drug resistance
Candida Biofilm Resistance
• 50% of all hospital infections are associated with medical devices. These infections invariably involve biofilm growth
• The most important modifiable factor associated with reduced mortality in patients with candidemia is catheter removal (N=1915, Mortality 28% removed vs 41% retained)
• Drug treatment of Candida biofilms is largely ineffective, primarily due to drug resistance
Drug (µg/ml) Biofilm-Associated
Resistance (Biofilm EC50/Plank MIC)
Amphotericin B 6-100x
Anidulafungin 8-32x
Fluconazole >1000x
Flucytosine >1000x
Baillie et al. 1998, Andes in press, Mukherjee et al. 2003
MIC=minimal inhibitory concentration EC50 (Effective concentration) is the drug concentration associated with a 50% reduction
• Candida mechanisms considered • Learning from prokaryotes • Candida matrix composition and
resistance contribution • Genetic basis for matrix associated
resistance • Proposed Model • Current studies
Candida Biofilm Resistance Overview
• Pump mutants are more susceptible, but a large amount of resistance phenotype remains unexplained
Impact of Efflux Pumps
Strain Planktonic MIC
Biofilm MIC
3153a 4 >256 Δcdr1 0.25 128 Δcdr2 0.25 128 Δcdr1/Δcdr2 0.25 256 Δmdr1 0.25 64 Δcdr1/Δmdr1 0.25 256
Ramage et al JAC 2002;49:973
Uppuluri et al AAC 2008;52:1127
Role of Calcineurin Pathway
• Pharmacologic and genetic inhibition of the pathway enhances triazole biofilm susceptibility
Impact of Growth Rate
Rate of growth controlled by glucose content of media (lower concentration = slower growth) l Planktonic cells n o ¡ Biofilm cells
AmB, Fluc, and 5FC added at 20X planktonic MIC = no impact on biofilm viability regardless of growth rate
Ballie and Douglas AAC 1998;42:1900
Persister Phenotype
• Persister cells exhibited resistance to AmB • Upregulation of glucan genes and down regulation of
sterol genes
Khot et al 2006;50:3708
Impact of Matrix - Penetration
Biofilm on filter disc
Fresh filter disc
Antifungal containing agar
Candida containing agar
Zone of inhibition
Fresh filter disc
Zone of inhibition correlated with standard curve from Antifungal spiked filter discs
Al-Fattini and Douglas AAC 2004:48:3291
Impact of Matrix - Penetration
C = biofilm diameter C0 = spiked disc diameter Each symbol represents different Candida species After 1 h the penetrated concentration exceeded the planktonic MIC many fold
Al-Fattini and Douglas AAC 2004:48:3291
Impact of Amount of Matrix
MORE MATRIX LESS MATRIX
• Reduced susceptibility to AmB associated with increase in matrix
Al-Fattani et al J Med Microbiol 2006;55:999
• P. aeruginosa biofilm drug resistance
- Cyclic β-1,3 glucan in the periplasm sequesters antibiotic
• rapA influence on E. coli biofilm resistance. – Decreased mutant matrix
carbohydrate
Mah et al Nature 2003;426:306, Lynch et al. Antimicrob Agents Chemother. 2007
Bacterial Glucan-Mediated Drug Resistance
Cell Wall β-1,3 glucan Content From Biofilm and Planktonic Cells
Nett et al. Antimicrob Agents Chemother 2007;51:510
Cell Growth Condition
Biofilm Log Stationary
Mic
rogr
ams
0
200
400
600
800
1000
1200
1400
1600
Alkali soluble glucanB 1,6 glucanB 1,3 glucanB 1,3 and 1,6 glucanTotal carbohydrate
Biofilm Planktonic2 um
A
1 um
B
Biofilm Planktonic2 um
A
2 um
A
1 um
B
1 um
B
Biofilm
200 nm
260 nm
Planktonic
120 nm
200 nm
Extracellular Matrix Composition Carbohydrate 41% Phosphorus 0.4% Glucose 15.9% Protein 5.2% Hexosamine 3.4%
Ballie and Douglas JAC 2000;46:397
In vitro Supernatant
In vitro Matrix
In vivo Serum
Beta-‐1,3 glucan (pg/ml)
0
50
100
150
200
250
300 Biofilm
Non-‐Biofilm
*
* *
Candida Matrix Composition
• Increased Beta 1,3 glucan, compared to planktonic cells both in vivo and in vitro
Nett Antimicrob Agents Chemother. 2007 ;51:510
Biofilm:Antifungal Association
- Using radio-labeled fluconazole, the majority of fluconazole associating with the intact biofilm was found in the biofilm matrix component
Nett et al. J Infect Dis. 2010
0500
1000150020002500300035004000
H3
Fluc
onaz
ole
(CPM
)
Inta
ctB
iofil
m
Bio
film
Mat
rix
Cel
l Wal
l
Cyt
opla
sm
- In vitro combination assays found disruption of glucan by β 1,3 glucanase treatment rendered biofilms more susceptible to fluconazole,
- Disruption of glucan by β 1,3 glucanase decreased the amount of radio-labeled fluconazole associating with the intact biofilm and the biofilm matrix *p<0.05
Impact of Glucan Modification on Fluconazole Susceptibility In vitro
Nett et al. Antimicrob Agents Chemother. 2007, Nett et al. J Infect Dis. 2010
50%
100%
0 125
Fluconazole (µg/ml)
Bio
film
Gro
wth
(%)
Control
B 1,3 glucanase
C. albicans Biofilm Gene Expression
114 > 1.5 fold at 24 h 111 > 1.5 fold at 12 h 82 same genes
Nett et al J Infect Dis 2009
ORF Gene Putative Function 19.2495 FKS1 β-1,3 glucan synthase 19.2929 GSL2 Glucan synthase
19.5058 SMI1 β-1,3 glucan synthesis 19.3829 PHR1 β-1,3 glucanosyltransferase 19.4565 BGL2 β-1,3 glucosyltransferase 19.2990 XOG1 β-1,3 glucanase 19.2237 SPR1 β-1,3 glucosidase 19.6276 GDB1 Glucanotransferase 19.1719 SGA1 Glucosidase 19.4668 SCW11 β-1,3 glucosidase 19.7434 GLG2 Glucosyltransferase
Role of FKS1 in C. albicans Biofilm Resistance to Fluconazole In vitro
Nett et al. J Infect Dis. 202;171:2010
- Modulation of FKS1 impacts biofilm resistance to fluconazole *p<0.05
Bio
film
Gro
wth
50%
100%
0 15 7.5 1
Doxycycline (ng/ml) Inhibition of FKS1
Control
Fluconazole 62.5 µg/ml
* *
* * 50%
100%
Reference FKS1/fks1∆ FKS1-S645F
Control Fluconazole 15.6 µg/ml Fluconazole 250 µg/ml
Fluconazole Dose (mg/kg/12 h)!3! 10! 30!
Cha
nge
Log1
0 C
FU/!
Kid
neys!
-2"
-1"
0"
1"
Reference strain!
FKS1/fks1∆!
Nett et al J Infect Dis 202;171:2010
Log1
0 C
FU/C
athe
ter!
Reference"
FKS1/fks1∆"
4"
5"
6"
7"
NaCl !Control"
Fluconazole!250 μg/ml"
*!
Biofilm Catheter Disseminated – No Biofilm
Planktonic – In vitro Strain MIC (mg/l)
WT 0.5 FKS1 -/+ 0.5
Role of FKS1 in C. albicans Biofilm Resistance In vivo and for Planktonic Cells
Reference FKS1/&s1∆ TDH3-‐FKS1
Impact of FKS1 Modulation on C. albicans Biofilm β-Glucan Production and Drug Sequestration
Ne5 et al. J Infect Dis 202;171;:2010
Reference FKS1/&s1∆ TDH3-‐FKS1
Glucan (pg)/serum
(ml)
In vitro-‐ Matrix
In vitro-‐ Supernatant
Glucan (pg)/Biofilm cells
(106)
0
200
400
500
1000
*
In vivo-‐ Serum
*
* *
*
[H3 ] Flucona
zole CPM
/biofi
lm weight (m
g)
0
20000
40000
60000
80000
100000
Intact Biofilm Matrix
*
*
*
*
* *
0
20000
40000
60000
80000
100000
*
*
*
*
* *
Reference FKS1/&s1∆ FKS1-‐S645F TDH3-‐FKS1
- Modulation of FKS1 influenced biofilm matrix glucan production in vitro and in vivo *p<0.05
- FKS1 modulation impacted association of radio-labeled fluconazole with biofilm matrix *p<0.05
- Less matrix was visualized in the FKS1/fks1∆ biofilm and more was seen in the TDH3-FKS1 biofilm growing on a rat venous catheters
scanning electron micrographs obtained at 5,000x measurement bars represent 1 µM
Biofilm Glucan Matrix and the Cell Wall Integrity Pathway?
S. cerevisae (Candida)
Increased Biofilm
Susceptibility?
Reduced Matrix Glucan?
Pkc1 N N
Bck1 N N
Mkk2 N N
Slt2 (Mkc1) N N
Knr4 (Smi1) Y Y
Rlm1 Y Y
Fks1 Y Y
Interaction of Glucan matrix and CWI Pathway, but different triggers and upstream control
Fks1p
Smi1p
Rlm1p
Fluconazole Resistance and SMI1 in an in vitro Candida biofilm model
• Deletion of smi1 modulates susceptibility to a drugs from a variety of classes, including fluconazole, amphotericin B and anidulafungin • Planktonic susceptibility to these drugs was not impacted. • Fluconazole binding efficiency of the smi1 biofilm and matrix was decreased
Fluconazole (µg/ml)
Perc
ent R
educ
tion
25%
50%
75%
0 250 125
* *
Reference smi1∆/smi1∆ smi1∆/smi1∆ + pSMI1
CP
M/N
orm
aliz
ed B
iofil
m (1
0exp
)
0
20
40
60
80
100
Reference smi1/smi1
Intact Biofilm Matrix
SMI1 Impact on Biofilm Formation and Matrix
200
400
600
800
1000
Biofilm Matrix
Reference
SMI1/smi1∆
smi1∆ /smi1∆
Β 1
,3 g
luca
n (p
g/m
l)
Regulation of Glucan Synthesis by SMI1
• Disruption of SMI1 diminishes transcription of FKS1 • Overexpression of FKS1 in an smi1Δ/smi1Δ background restores resistance to antifungal
Transcript
Nor
mal
ized
Exp
ress
ion
Rat
io
A
SMI1 FKS1
0.2
0.4
0.6
0.8
1
1.2 Reference
SMI1/smi1∆
smi1∆/smi1∆
B
Fluconazole (µg/ml)
Perc
ent r
educ
tion
10%
20%
30%
40%
50%
0 125 500
smi1∆/smi1∆
smi1∆/smi1∆ +TDH3-FKS1
**
Impact of FKS1 Modulation on C. albicans Biofilm Cell Wall and Susceptibility to
Stress Inducing Agents FKS1/fks1∆ TDH3-FKS1 Reference
TET-FKS1 +Doxycyline
TET-FKS1 - Doxycycline FKS1-S645F
Strain
Growth Response to Cell Stress (MIC or EC50)
37oC 42oC NaCl (M)
H2O2 (mM)
Congo Red
(µg/ml)
EtOH (%)
SDS (%)
Planktonic Reference ++ ++ 2 16 0.6 3.1 0.03
FKS1/fks1∆ + + 2 8 0.6 1.5 0.03
Biofilm Reference + ND >2 125 62.5 25 0.035
FKS1/fks1∆ + ND >2 125 62.5 25 0.035
Nett et al J Infect Dis 202;171:2010
Interaction of AmB with C. albicans Biofilms and Glucan
Vediyappan et al AAC 2010;54:2096
AmB bound to biofilm matrix
AmB bound to glucan
Impact of FKS1 Modulation on C. albicans Biofilm Drug Resistance to Amphotericin B
Amphotericin B (µg/ml) 0
Bio
film
Gro
wth
2 1 0.5 0.25 0.13 0.06
*
* *
0%
25%
50%
75%
100% *
*
Reference
FKS1/fks1∆ TDH3-FKS1
- The TDH3-FKS1 strain was more resistant to amphotericin B *p<0.05 - The FKS1/fks1∆ biofilm was more susceptible to amphotericin B *p<0.05 - Planktonic MICs were not significantly different among the strains
- Similar impact on two additional drug classes, flucytosine and anidulafungin
Nett et al Antimicrob Agents Chemother. 2010
chitin chitin
XX
chitinchitinchitin chitinchitin
XX
Fks1pPlasma MembraneMatrix GlucanFluconazoleBiofilm Cell Wall
Fks1pPlasma MembraneMatrix GlucanFluconazoleBiofilm Cell Wall
Hypothesized Genetic Control of C. albicans Biofilm Resistance
Antifungal
?
Current Studies
• We are near completion of refined matrix composition analysis
• We are exploring further components of the glucan matrix pathway to identify control points and potential drug targets
• We have identified and are refining analysis of genes important for matrix delivery or deposition
Acknowledgements Lab Members Jacque Bohrmueller Mike Cain Kyler Crawford Alex Lepak Karen Marchillo Jeniel Nett Kelly Ross Hiram Sanchez Heather Taff Robert Zarnowski
Collaborators Joe Heitman Bruce Klein Aaron Mitchell Mahmoud Ghannoum Leah Cowen Geraldine Butler Sandy Johnson Clarissa Nobile
Funding Sources NIH, VA, Mycoses Study Group Astellas, Pfizer, Merck
Impact of FKS1 Modulation on C. albicans Biofilm Drug Resistance to Flucytosine
Flucytosine (µg/ml)
0%
25%
50%
75%
100%
0 8 4 2 1 0.5 0.25
*
* * * *
*
-The FKS1/fks1∆ biofilm and the TET-FKS1 biofilm under doxycycline repression of FKS1 were more susceptible to flucytosine -Planktonic MICs were not significantly different among the strains
- Similar impact on two additional drug classes, amphotericin and anidulafungin
Flucytosine (µg/ml)
0
25%
50%
75%
100%
0 1 2 4 8
No Doxycycline
Doxycycline 7.5 ng/ml
Bio
film
Gro
wth
Reference FKS1/fks1∆ TDH3-FKS1
Nett et al Antimicrob Agents Chemother. 2010
Anidulafungin (µg/ml)
Impact of FKS1 Modulation on C. albicans Biofilm Drug Resistance to Anidulafungin
Bio
film
Gro
wth
0%
25%
50%
75%
100%
0 0.12 0.06 0.03 0.02 0.01 0.004
*
*
Anidulafungin (µg/ml) 0 0.002 0.004 0.01 0.02
0
25%
50%
75%
100%
-The FKS1/fks1∆ biofilm and the TET-FKS1 strain under doxycycline repression of FKS1 were more susceptible to anidulafungin *p<0.05 -The TDH3-FKS1 biofilm was significantly more resistant to anidulafungin *p<0.05 Planktonic MICs were not significantly different for each strain
Reference FKS1/fks1∆ TDH3-FKS1
No Doxycycline
Doxycycline 7.5 ng/ml
Nett et al Antimicrob Agents Chemother. 2010