interactions Françoise Van Bambeke, PharmD, PhD Paul M. Tulkens, MD, PhD

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1 Antiinfectives and biofilms interactions Françoise Van Bambeke, PharmD, PhD Paul M. Tulkens, MD, PhD Pharmacologie cellulaire et moléculaire Louvain Drug Research Institute Université catholique de Louvain, Brussels, Belgium < avril 2018 Port El Kantaoui, Sousse, Tunisie Antiinfectives and biofilms 1

2 Antiinfectives and biofilms 2 Microbiological difficulties and challenges in anti-infective therapy Resistance Persistence Small Colony variants Intracellular forms Biofilms

3 Antiinfectives and biofilms 3 Difficultés et défis microbiologiques de l'antibiothérapie Resistance Persistence Small Colony variants Intracellular forms Biofilms

4 Antiinfectives and biofilms 4 Difficultés et défis microbiologiques de l'antibiothérapie Resistance Persistence ciprofloxacin gentamicin Small Colony variants oxacilllin vancomycin Intracellular forms Biofilms Johnson & Levin. PLoS Genet. 2013;9:e PMID: ; Not all bacteria are dead!

5 Antiinfectives and biofilms 5 Difficultés et défis microbiologiques de l'antibiothérapie Resistance Persistence Small Colony variants Intracellular forms Biofilms Proctor et al. Nat Rev Microbiol 2006;4: PMID: Eradication of 'small colony variants" requires prolonged antibiotic treatments often in multi-drug combinations

6 Antiinfectives and biofilms 6 Difficultés et défis microbiologiques de l'antibiothérapie Resistance S. aureus in human osteoblasts Persistence Small Colony variants Intracellular forms Biofilms Kalinka et al., Int J Med Microbiol. 2014; 304: PMID: Recalcitrant to eradication.

7 Antiinfectives and biofilms 7 Difficultés et défis microbiologiques de l'antibiothérapie Résistance Persistance Small Colony variants Françoise Van Bambeke Formes intracellulaires Wafi Siala Biofilms

8 Antiinfectives and biofilms 8 Biofilms in human infections Biofilms are associated to 65 a -80 b % of human infections and can colonize virtually all organs ear nose throat mouth & teeth eye lung heart kidney gall bladder pancreas nervous system skin bone *** implanted medical devices a CDC 1999; b Lewis et al, Nat Rev Microbiol. 2007; 5:48-56

9 Antiinfectives and biofilms 9 Antibiotics and biofilms in clinical practice Treatment failure is not rare

10 Antiinfectives and biofilms 10 In vitro static models pegs multiwell plates

11 Antiinfectives and biofilms 11 Antibiotic activity: planktonic vs. biofilm cultures Macià et al, Clin Microbiol Infect. 2014; 20(10):981-9

12 Antiinfectives and biofilms 12 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:

13 Antiinfectives and biofilms 13 Comparing antibiotic activity: planktonic / biofilm cultures Ampicillin and levofloxacin vs. H. influenzae from middle ear fluid MIC MIC MBC MBC MBEC MBEC Activity against biofilm << activity against planktonic bacteria Takei et al, J Infect Chemother 2013; 19:504 9

14 Antiinfectives and biofilms 14 PK/PD studies: the principles Pharmacokinetics 0.4 conc. vs time Pharmacodynamics conc. vs effect Conc. Effet Temps Conc. (log) 10-3 Effet 1 PK/PD effect vs time 0 0 Temps

15 Antiinfectives and biofilms 15 Static models: 96-well polystyrene plates appropriate dyes to evaluate biomass or bacterial load

16 Antiinfectives and biofilms 16 Quantifying biomass and metabolic activity in biofilms biofilm mass crystal violet Christensen et al, Infect. Immun. 1982; 37:318 26

17 Antiinfectives and biofilms 17 Quantifying biomass and metabolic activity in biofilms biofilm mass crystal violet Christensen et al, Infect. Immun. 1982; 37: Gram(+) bacteria metabolic activity Gram(-) bacteria resazurin fluorescein diacetate resorufin fluorescein Tote et al, 2008; Lett. Appl. Microbiol. 46: Wanandy et al, J Microbiol Methods 2005;60:21-30

18 CFU counting vs. RF fluorescence An example for S. aureus relation between fluorescence and bacterial inoculum for S. aureus log CFU/ml log OD 620 nm min. incubation log resorufin fluorescence -2.5 CFU & RF signal proportional sensitivity depending on incubation time Bauer, Siala et al, Antimicrob Ag Chemother. 2013;57: ; Van den Driessche et al, J.Micr. Meth. 2014; 98: Antiinfectives and biofilms 18

19 Pharmacodynamic model for antibiotic activity An example with young biofilm of S. aureus vancomycin % control value C «rel. potency» CV RF 20 0 CT log 10 concentration (X MIC) Emax «efficacy» Bauer, Siala et al, Antimicrob Ag Chemother. 2013;57: Antiinfectives and biofilms 19

20 Pharmacodynamic model for antibiotic activity Comparison between young and mature biofilms vancomycin vs. young biofilms vancomycin vs. mature biofilm % control value % control value CV RF 20 CV RF 0 CT log 10 concentration (x MIC) 0 CT log 10 concentration (x MIC) Bauer, Siala et al, Antimicrob Ag Chemother. 2013;57: Antiinfectives and biofilms 20

21 Antiinfectives and biofilms 21 S. pneumoniae biofilms - influence of maturity moxifloxacin 120 metabolic activity biomass % control value day 11 day Log 10 concentration (X MIC) Log 10 concentration (X MIC) maximal efficacy with maturity Vandevelde et al, Antimicrob Ag Chemother. 2014; 58:

22 Antiinfectives and biofilms 22 S. pneumoniae biofilms - influence of maturity moxifloxacin 120 metabolic activity biomass % control value day 11 day Log 10 concentration (X MIC) Log 10 concentration (X MIC) relative potency with maturity Vandevelde et al, Antimicrob Ag Chemother. 2014; 58:

23 Antiinfectives and biofilms 23 Comparison of PD parameters for different drugs S. pneumoniae Vandevelde et al, Antimicrob Ag Chemother. 2014; 58:

24 Antiinfectives and biofilms 24 How to explain this apparent resistance or tolerance? PK/PD 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,..])

25 Antiinfectives and biofilms 25 PK/PD parameters 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

26 S. aureus biofilms Importance of antibiotic concentration inside biofilms for activity Activity in biofilm is correlated to antibiotic penetration Siala et al, Antimicrob. Ag. Chemother. 2014, 58: Antiinfectives and biofilms 26

27 How to help antibiotic to reach their target? Disrupt the biofilm matrix Rabin et al., Future Med. Chem. 2015; 7: Antiinfectives and biofilms 27

28 Antiinfectives and biofilms 28 IacA and polysaccharide synthesis in S. aureus Ica A is involved in N-acetylglucosamine homopolymer synthesis Otto et al., Nat. Rev. Microbiol. 2009; 7:555-67

29 S. aureus biofilms Importance of icaa expression and PNAG abundance for antibiotic activity in biofilms strain icaa expression ATCC S ± 0.5* 2003/ ± 0.6 * 2009S ± 0.5* 2005/ ± 0.4* 2005/ ± 0.9* 2009S ± 0.2* 2003/ ± 0.7* log 2 C 25 moxifloxacin (mg/l) in combination with caspofungin ATCC / S / / S S /651 r: icaa relative expression log 2 C 50 delafloxacin (mg/l) in combination with caspofungin ATCC / S / / S S /651 r: icaa relative expression Fluoroquinolone activity in biofilm is inversely correlated with icaa expression Siala et al, Nature Communications 2016; 7: Antiinfectives and biofilms 29

30 The antifungal caspofungin as an inhibitor of polysaccharide synthesis Glucan synthase Candida albicans Atkin et al, FEBS Lett. 2014;588: Adapted from Arnold, Kucer s 6the edition Siala et al, Nature Communications 2016; 7: Antiinfectives and biofilms 30

31 Inhibition of IcaA by caspofungin increases fluoroquinolone penetration in biofilms S. aureus biofilms ATCC /651 UDP conc (µm) Log 10 CAS conc (mg/l) CAS reduces PNAG in the matrix CAS inhibits IcaA activity CAS increases fluoroquinolone concentration in biofilms Siala et al, Nature Communications 2016; 7: Antiinfectives and biofilms 31

32 Caspofungin increases fluoroquinolone activity in vitro and in vivo Catheters in vitro Siala et al, Nature Communications 2016; 7: Antiinfectives and biofilms 32

33 Caspofungin increases fluoroquinolone activity in vitro and in vivo Catheters in vitro Catheters in vivo Siala et al, Nature Communications 2016; 7: Antiinfectives and biofilms 33

34 Antiinfectives and biofilms 34 PK/PD parameters in biofilms nutrients & oxygen catheter, bone, skin, cardiac valve, pharmacodynamics bacterial responsiveness (metabolic activity of bacteria) antibiotic expression of activity (local environment [O 2, ph,..])

35 Environmental ph S. aureus + delafloxacin 7 5 HO F N N O N COOH F H 2 N F delafloxacin * Labelling with Seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) Correlation between delafloxacin relative potency and MIC at the ph of the surface of the biofilm Biofilm ph may influence antibiotic intrinsic activity Siala et al, Antimicrob. Ag. Chemother. 2014, 58: Antiinfectives and biofilms 35

36 Antiinfectives and biofilms 36 In vitro dynamic models permanent fluid stirring unidirectional flow replacement constant conditions

37 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 2012;7(11):e Antiinfectives and biofilms 37

38 Dynamic models: bioreactors CDC reactor: Mimicking the pharmacokinetic profile of selected antistaphylococcal agents fusidic acid theor.: 0.04 h -1 exper.: 0.14 ± 0.08 h -1 daptom ycin theor.: 0.09 h -1 exper.: 0.07 ± 0.02 h -1 concentration (m g/l) concentration (m g/l) tim e (h ) tim e (h ) lin e z o lid theor.: 0.12 h -1 exper.: 0.28 ± 0.08 h -1 vancom ycin theor.: 0.12 h -1 exper.: 0.12 ± 0.06 h -1 concentration (m g/l) concentration (m g/l) tim e (h ) tim e (h ) Siala et al, Antimicrob. Agents Cheomther. in press Antiinfectives and biofilms 38

39 Dynamic models: bioreactors CDC reactor: Activity of antibiotics alone or in combination CTRL FUS A T C C Antibiotics alone inactive in this model! VAN DAP LDZ FUS+VAN FUS+DAP FUS+LDZ 8 lo g 10 CFU/cm conditioning phase drug phase tim e (h ) Siala et al, submitted Antiinfectives and biofilms 39

40 Antiinfectives and biofilms 40 Conclusion: PK/PD in biofilms: what did we learn? 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

41 Antiinfectives and biofilms 41 Pros and Cons on biofilms models Coenye et al, Clin Microbiol Infect pii: S X(18) doi: /j.cmi

42 Acknowledgments Hariri Mustafa Yvan Diaz Iglesias Sona Kucharíková Patrick Van Dijck Nathalie Vandevelde Wafi Siala Antiinfectives and biofilms 42