Anti-staphylococcal activity of antibiotics in biofilm and host cell

Transcription

1 Anti-staphylococcal activity of antibiotics in biofilm and host cell Françoise Van Bambeke, PharmD, PhD Pharmacologie cellulaire et moléculaire Louvain Drug Research Institute Université catholique de Louvain, Brussels, Belgium < 1

2 Persistent forms of infection by S. aureus BACTERIUM HOST ANTIBIOTIC catheter, bone, skin, cardiac valve, intracellular infection biofilm 2

3 Persistent forms of infection by S. aureus and antibiotics PK parameters: Access and accumulation at the infection site PD parameters: Expression of antibiotic activity Bacterial responsiveness Cooperation with the host BACTERIUM HOST ANTIBIOTIC catheter, bone, skin, cardiac valve, intracellular infection biofilm 3

4 Intracellular infection Nguyen et al, AAC (29) 53:

5 PK/PD parameters and intracellular activity metabolism binding cooperation with host defenses influx efflux accumulation and bioavailability bacterial responsiveness physico-chemical conditions Carryn et al, Infect Dis Clin North Am (23) 17:

6 In vitro model of intracellular infection Opsonization Incubation with 1 % human serum in culture medium Phagocytosis Incubation of bacteria with cells at an appropriate MOI Extracellular wash Elimination of non phagocytosed bacteria by incubation with 5-1 X MIC gentamicin; elimination of gentamicin by washing Gentamicin (G) G G G G G G G G G G G G Determination of residual intracellular inoculum Determination of cfu and protein content in cell lysates cfu proteins Intracellular infection Incubation over time in control conditions or with antibiotics Antibiotic (AB) AB AB AB AB AB AB Buyck et al (216) ISBN

7 In vitro model of intracellular infection remains in vacuoles Seral et al, AAC (23) 47:

8 Setting-up appropriate models for the study of cellular activity of antibiotics moxifloxacin & S. aureus log CFU 24h-h intra extra log extracellular concentration (mg/l) Barcia-Macay et al, AAC (26) 5:

9 Setting-up appropriate models for the study of cellular activity of antibiotics moxifloxacin & S. aureus log CFU 24h-h intra extra log extracellular concentration (mg/l) model C stat (x MIC) extra.27 intra.63 Barcia-Macay et al, AAC (26) 5: relative potency 9

10 Setting-up appropriate models for the study of cellular activity of antibiotics moxifloxacin & S. aureus log CFU 24h-h intra extra log extracellular concentration (mg/l) model C stat (x MIC) E max extra (5.22 to 2.51) intra (3.31 to 2.22) Barcia-Macay et al, AAC (26) 5: relative potency maximal efficacy 1

11 What do these parameters tell you? log CFU 24h-h intra extra log extracellular concentration (mg/l) relative potency Estimation of the concentration needed to reach a specified effect Measure of the «intracellular MIC» «PK-related» parameter: accumulation in the infected compartment intracellular bioavailability influence of local environment on intrinsic activity ph oxidant species In most cases Cs intra Cs extra 11

12 What do these parameters tell you? log CFU 24h-h intra extra log extracellular concentration (mg/l) maximal efficacy Estimation of the maximal reduction in inoculum for an infinitely large concentration Measure of the killing capacity «PD-related» parameter mode of action of the drug bacterial responsiveness cooperation with host defenses In most cases Emax intra <<< Emax extra 12

13 Antibiotic accumulation and subcellular distribution diffusion possible re-distribution confined in vacuoles endocytosis linezolid: ~ 1 x lincosamides: 1-4 x tetracyclines: 2-4 x rifampin : 2-1 x synercid: 3-5x β-lactams; fast; ~ 1 x fluoroquinolones : fast CIP, LVX : 4-1 x MXF, GAR, GMF : 1-2 x? aminoglycosides: slow ; 2-4 x glycopeptides: slow VAN ~ 8 x TLV ~ 5 x ORI ~ 15-3 x macrolides: fast ERY: 4-1 x CLR, ROX, TEL: 1-5x AZM, SOL: > 5 x some oxazolidinones: fast RDZ : 1 x mainly in vacuoles slow release diffusion/ segregation 13

14 Can we predict the intracellular activity based on intracellular concentrations? No correlation between intracellular concentration and intracellular activity Adapted from Van Bambeke et al., Curr. Opin. Drug Discov. Devel (26) 9:

15 Importance of accumulation/bioavailability Fluoroquinolones against L. monocytogenes vs. S.aureus intracellular bioavailability? log CFU from time L. monocytogenes GMF MXF CIP S aureus GMF MXF CIP ng/mg cell prot CIP MXF GMF fluoroquinolone Vallet et al, IJAA (211) 38: cell. concentr. (x MIC) to reach the target effect log extracell. concentr. (x MIC) log -2 log CIP MXF GMF L. monocytogenes target effect (difference from initial inoculum) log extracell. concentr. (x MIC) CIP MXF GMF S. aureus log -1. log target effect (difference from initial inoculum)

16 Importance of subcellular distribution Moxifloxacin & oritavancin against L. monocytogenes vs. S.aureus control moxifloxacin (4 mg/l) L. monocytogenes oritavancin (25 mg/l) S. aureus 2 2 MXF ORI log CFU from time time (h) time (h) AB needs to have access to the infected compartment adapted from Carryn et al, AAC (22) 46: Van Bambeke et al, AAC (24) 48: Barcia-Macay et al, AAC (26) 5:

17 Importance of bacterial phenotype comparison : isogenic strains with different phenotypes Krebs cycle oxidative stress SCV vs normal phenotype Normal Phenotype Spontanous revertants SCV Thymidine dependent DHPP+PABA DHP DHPS XTS DHFR CH 2 =THF DHF THF dump metabolism SMX Thy-dep SCV dtmp thiamine-pp TMP DNA Men-dep SCV X menadione menadione biosynthetic enzymes DEATH! Antioxidant pathway? NADH and FADH 2 menaquinone cytochromes ROS bactericidal antibiotic cell wall and protein synthesis ATP electrochemical gradient Haemin-dep SCV hemin X biosynthetic enzymes haemin Cytochrome oxidase proton motive force F F 1 ATPase carotenoid synthesis growth cell wall active antibiotics aminoglycoside transport pigment synthesis Vergison et al, JAC (27) 59:893-9 Garcia et al, JAC (213) 68:

18 Importance of bacterial phenotype comparison : isogenic strains with different phenotypes Efficacy lower against SCV, possibly related to slower metabolism? Nguyen et al, AAC (29) 53:

19 Antibiotic combinations FME synergy additivity indifference antagonism antagonism antagonism drug A drug B.3.1 Nguyen et al. AAC (29) 53: Combinations synergism! 19

20 Influence of intracellular ph 2 MRSA are as susceptible as MSSA to β-lactams when intracellular! Lemaire et al., AAC (27) 51:

21 21 Influence of intracellular ph MRSA are as susceptible as MSSA in broth at acidic ph Lemaire et al., AAC (27) 51:

22 PBP2a conformation is modified by acidic ph PBP2a OXA PBP2a OXA closed! open! Lemaire et al., J Biol Chem (28) 283:

23 Importance of cell defense mechanisms comparison : isogenic strains with different phenotypes log CFU from time control WT mend mends hemb hembgc -1 cloxacillin N-acetylcystein WT mend mends hemb hembgc -1 cloxacillin The menadione SCV is hypersusceptible intracellularly because of the combined effect of acidic ph and oxidant species log extracellular concentration (mg/l) cloxacillin ph 7.4 control + H 2 O 2 cloxacillin ph 5.4 M IC (m g/l) WT mend mends hemb hembs hembgc WT mend mends hemb hembs hembgc strains strains Garcia al, JAC (212) 67:

24 Comparison of a single antibiotic towards several strains comparison : moxifloxacin against increasingly resistant MRSA MIC (mg/l) NRS192 KKH II-7924 SA1 NRS386 SA69 HMC 551 NRS384 SA481 log 1 cfu/mg protein (24 h - h) log 1 extracellular concentration (mg/l) log 1 extracellular concentration (x MIC) Cs ~ MIC Lemaire et al, JAC (211) 66:

25 Comparison of several antibiotics towards a single strain Relative potency? effect conc. Maximal efficacy? effect conc. 8 4 MIC 3 2 extra intra Static concentration (mg/l) linezolid vancomycin clarithromycin quin.-dalf. penicillin G Cs ~ MIC gentamicin telithromycin moxifloxacin rifampicin log cfu (24 h - h) None TMP-SMX AZI TC TGC VAN CLX LZD CLI CIP RIF DAP MXF Q-D TLV ORI Emax intra << Emax extra Lemaire et al (29) - ISBN:

26 From in vitro to in vivo : The mouse peritonitis model Electron microscopy of peritoneal fluid post infection with S. aureus Extracellular S. aureus Intracellular S. aureus Sandberg et al, AAC (29) 53:

27 in vitro vs in vivo in vitro (macrophages) Linezolid & S. aureus in vivo (peritonitis) Sandberg et al, JAC (21) 65:

28 Antibiotics and intracellular S. aureus: take home message 28 Simple equation? Intracellular activity =X MIC x accumulation

29 Antibiotics and intracellular S. aureus: take home message 29 shopping list high intracellular bioavailability capacity to rejoin the infected compartment not substrate for efflux pumps low MIC at both neutral and acidic ph highly bactericidal, including against slow growing bacteria no cell toxicity cooperation with cell defense mechanisms

30 Biofilms 3 University of Gothenburg

31 PK/PD parameters and activity in biofilms nutrients & oxygen catheter, bone, skin, cardiac valve, 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 [O 2, ph,..]) 31

32 PD parameters: planktonic vs. biofilm cultures Macià et al, Clin Microbiol Infect. (214) 2:

33 In vitro 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 (21) 22:

34 PD parameters: planktonic vs. biofilm cultures Ampicillin and levofloxacin vs. H. influenzae from middle ear fluid MIC MIC MBC MBC MBEC MBEC slowly bactericidal antibiotic: MBEC >> MBC >>MIC rapidly bactericidal antibiotic: MBEC > MBC ~ MIC Takei et al, J Infect Chemother (213) 19:

35 Dynamic models: bioreactors CDC reactor: constant mixing by stirring kinetic experiments with change in medium composition over time high shear stress drip flow reactor ; low shear forces Stewart et al, PLoS One (212) 7:e556 35

36 Antibiotic activity - mimicking human exposure DAP measured vs theorical LDZ measured vs theorical LDZ theoretical D A P conc (m g/l) 1 5 DAP theoretical DAP measured LDZ conc (mg/l) LDZ measured Tim e (h) Tim e (h) FUS conc (mg/l) FUS measured vs theorical 5 FU S theoretical Tim e (h) FUS measured V A N conc (m g/l) VAN measured vs theorical 3 VAN theoretical Tim e (h) VAN measured Siala et al, Biofilm Resistance, 216 antibiotic fcmax (mg/l) fcmin (mg/l) k calc /t 1/2 (h -1 )/(h) k measured (h -1 ) DAP (8h).7 ±.2 VAN (6h).12 ±.6 LZD (6h).28 ±.8 FUS (6h).15 ±.8 36

37 Antibiotic activity - mimicking human exposure ATCC25923 CTRL VAN DAP LDZ FUS FUS+VAN FUS+DAP FUS+LDZ log 1 CFU/ml conditioning phase 3.5 Drug phase tim e (h) Siala et al, Biofilm Resistance, 216 Combination much more active 37

38 In vitro models for PK/PD studies: 96-well polystyrene plates appropriate dyes to evaluate biomass or bacterial load 38

39 Quantifying biomass and metabolic activity in biofilms biofilm mass crystal violet Christensen et al, Infect. Immun. (1982) 37: metabolic activity resazurin resorufin Tote et al, Lett. Appl. Microbiol. (28) 46:

40 CFU counting vs. RF fluorescence relation between fluorescence and bacterial inoculum for S. aureus log CFU/ml log OD 62 nm min. incubation log resorufin fluorescence -2.5 CFU & RF signal proportional sensitivity depending on incubation time Bauer et al, AAC (213) 57: Van den Driessche et al, J.Micr. Meth. (214) 98:31 4 4

41 S. aureus model: growth kinetics 3 25 crystal violet absorbance resorufin fluorescence % of value at 6 h time of incubation (h) young biofilm mature biofilm 41

42 Pharmacodynamic model for antibiotic activity An example with young biofilm of S. aureus vancomycin 12 1 CV RF % control value C «rel. potency» 2 CT log 1 concentration (X MIC) Emax «efficacy» Bauer, Siala et al, AAC (213) 57:

43 S. aureus mature biofilms: comparison of drugs ATCC33591 (MRSA) vancomycin delafloxacin daptomycin % control value % control value % control value CT CV RF log 1 concentration (X MIC) 2 CT CV RF log 1 concentration (X MIC) 2 CT CV RF log 1 concentration (X MIC) life dead more active on viability than on matrix huge difference among drugs Bauer, Siala et al, AAC (213) 57:

44 Parameters affecting antibiotic activity in biofilms 2 clinical isolates of S. aureus- delafloxacin 12 viability 23/ /S27 biomass 23/ S27 12 Percentage of control value CT CT log 1 concentration (mg/l) What makes the difference? Siala et al, AAC (214) 58:

45 PK parameter: antibiotic penetration DFX / live / dead concentration in biofilm (% added concentration) S27 23/ biofilm depth (µm) Correlation antibiotic penetration potency in biofilms Siala et al, AAC (214) 58:

46 PK parameter: antibiotic penetration DFX / live / dead concentration in biofilm (% added concentration) S27 23/ biofilm depth (µm) antibiotic penetration depends on matrix polysaccharide content Siala et al, AAC (214) 58:

47 Matrix disorganisation Disorganisation of EPS increases antibiotic penetration Norspermidine reduced the concentrations (3 x) and the diameters (2 x) of exopolysaccharide supramolecular particles. Siala et al, AAC (214) 58:

48 PD parameter: environmental ph basic acidic Correlation MIC at biofilm ph potency in biofilms Siala et al, AAC (214) 58:

49 in vitro vs in vivo S27 23/ log 1 CFUs/catheter piece CTRL MXF 2.5 CTRL MXF treatm ent Siala et al, submitted for publication 49

50 in vitro vs in vivo control MXF DFX Xen36 Total Flux (p/s) control MXF DFX log 1 CFUs/catheter piece log 1 CFUs/catheter piece 4.x CTRL days Siala et al, unpublished MXF CTRL DFX 5

51 PD parameter: importance of metabolic state Coulon, Bioassays (214) 36:

52 Anti- persister and biofilms targets ClpP, core unit of a major bacterial protease complex. Synergistic with antibiotics in biofilms Enrichment in degraded proteins Coulon et al, Nature (213) 53:

53 Antibiotics and S. aureus biofilm: take home message 53 How do biofilms counter-act antibiotic activity? MATRIX BACTERIA Composition Metabolic state Biophysical properties Useful strategies? Destructing the matrix Waking-up dormant cells

54 Acknowledgments intracellular infection Paul Tulkens Cristina Seral Maritza Barcia Sandrine Lemaire Huang Anh Nguyen Pierre Baudoux Laetitia Garcia Frédéric Peyrusson Anne Sandberg Niels Frimodt-Möller Transparency declaration 54

55 Acknowledgments - biofilms Julia Bauer Wafi Siala Sona Kucharíková Patrick Van Dijck Transparency declaration 55

56 Acknowledgments other collaborations In Belgium O. Denis, Université libre de Bruxelles H. Rodriguez-Villalobos, Université catholique de Louvain Outside Belgium. B. Kahl, University of Muenster, Germany P. Appelbaum, Hershey Medical Center, PA S. Mobashery, Univeresity of Notre-Dame, IL 56

57 Acknowledgments 57