Antibiotic resistance by efflux: from molecular aspects to cinical impact

Transcription

1 University of Notre-Dame, Notre Dame, IN 1 Antibiotic resistance by efflux: from molecular aspects to cinical impact Françoise Van Bambeke & Paul M. Tulkens Pharmacologie cellulaire et moléculaire Louvain Drug Research Institute Université catholique de Louvain Brussels, Belgium Department of Chemistry and Biochemistry University of Notre Dame (College of Science), Notre Dame, IN 16 September 2013

2 University of Notre-Dame, Notre Dame, IN 2 Efflux is a Belgian specialty. Manneken-Pis Folon Magritte

3 Discovery and significance University of Notre-Dame, Notre Dame, IN 3

4 University of Notre-Dame, Notre Dame, IN 4 Chemotherapeutic agents exert toxic effects on specific target cells antibiotics antifungals anticancer agents

5 Chemotherapeutic agents exert toxic effects on specific target cells enzymes nucleic acids ribosomes How can these drugs reach their target inside the cells? Van Bambeke et al. (2000) Biochem. Pharmacol. 60: University of Notre-Dame, Notre Dame, IN 5

6 Reaching an intracellular target polar drug lipophilic drug physico-chemical properties are inadequate for reaching an intracellular target! Van Bambeke et al. (2000) Biochem. Pharmacol. 60: University of Notre-Dame, Notre Dame, IN 6

7 Reaching an intracellular target amphipathic drug most drugs are amphipathic by design, to be able to cross membrane barriers! Van Bambeke et al. (2000) Biochem. Pharmacol. 60: University of Notre-Dame, Notre Dame, IN 7

8 Intracellular chemotherapeutic agents But a diffusible compound may have potentially harmful effects! Van Bambeke et al. (2000) Biochem. Pharmacol. 60: University of Notre-Dame, Notre Dame, IN 8

9 Why efflux transporters? Extrusion by efflux pumps Van Bambeke et al. (2000) Biochem. Pharmacol. 60: University of Notre-Dame, Notre Dame, IN 9

10 Why efflux transporters? Extrusion by efflux pumps general mean of protection against cell invasion by diffusible molecules Van Bambeke et al. (2000) Biochem. Pharmacol. 60: University of Notre-Dame, Notre Dame, IN 10

11 Most antibiotics are amphiphilic! cationic amphiphiles fluoroquinolone macrolide tetracycline sulfamide rifampicin lincosamide Van Bambeke et al. (2000) Biochem. Pharmacol. 60: University of Notre-Dame, Notre Dame, IN 11

12 Most antibiotics are amphiphilic! anionic amphiphiles - -lactam - - fusidic acid fluoroquinolone Van Bambeke et al. (2000) Biochem. Pharmacol. 60: University of Notre-Dame, Notre Dame, IN 12

13 Antibiotic efflux transporters are ubiquituous spectrum narrow prokaryotes distribution Gram (+) Gram (-) eukaryotes MATE SMR MFS ABC broad RND Mesaros et al. (2005) Louvain médical. 124: University of Notre-Dame, Notre Dame, IN 13

14 Antibiotics as substrates of efflux pumps Antibiotic bacteria fungi superior class Gram (+) Gram(-) eucaryotes -lactams fusidic acid macrolides streptogramins tetracyclines aminoglycosides chloramphenicol rifamycins sulfamides trimethoprim fluoroquinolones Van Bambeke et al. (2000) Biochem. Pharmacol. 60: University of Notre-Dame, Notre Dame, IN 14

15 Antibiotic efflux and resistance: time line tetracyclines chloramphenicol -lactams macrolides fluoroquinolones rifampin aminoglycosides oxazolidinones University of Notre-Dame, Notre Dame, IN 15

16 University of Notre-Dame, Notre Dame, IN 16 Efflux: from molecular recognition to cellular impact

17 spectrum narrow Efflux as a mechanism of resistance in Gram-positive bacteria specific for one (or a few) families of drugs ABC MFS broad PatA/PatB of S. pneumoniae FQ, chl MsrA of S. epidermidis erythromycin NorA of S. aureus FQ,Tet, chl MefE of S. pneumoniae ML PmrA of S. pneumoniae FQ MefA of S. pyogenes ML University of Notre-Dame, Notre Dame, IN 17

18 FQ efflux pumps in S. pneumoniae S. aureus Primary transporters «ATP-Binding Cassette» PatA/PatB (Sp) Marrer et al, AAC 2006; 50: Secondary transporters (Proton motive force) PmrA (Sp) Gill et al, AAC 1999; 43:187-9 NorA (Sa) Gill et al, AAC 1999; 43:187-9 Terry et al., Nature Reviews Microbiology 2005; 3: University of Notre-Dame, Notre Dame, IN 18

19 spectrum narrow Efflux as a mechanism of resistance in Gram-negative bacteria specific for one (or a few) families of drugs MFS TetA of E. coli Tet MsrA of S. epidermidis erythromycin broad broad spectrum, conferring cross-resistance RND MexAB-OprM of P. aeruginosa -lac, FQ,Tet, ML, chl, rif, sulf AcrAB-TolC of E. coli -lac, FQ,Tet, ML, chl, rif, sulf University of Notre-Dame, Notre Dame, IN 19

20 Efflux and resistance in P. aeruginosa Constitutive basal expression overexpressed upon induction No basal expression; expression upon induction MexB MexY MexD MexF MexA MexX MexC MexE OprM OprM OprJ OprN Mesaros et al. (2005) Louvain médical. 124: University of Notre-Dame, Notre Dame, IN 20

21 Substrate specificity of efflux pumps 14 fluoroquinolones; Gram + versus Gram + MIC change [PatA+/-] (fold dilutions) S. aureus vs S. pneumoniae S. aureus vs S. pneumoniae R² = PEF GAR SPX DIF MXF LVX SAR FIN MAR ENX LOM GMF CIP NOR MIC change [NorA+/-] (fold dilutions) MIC change [PatB+/-] (fold dilutions) DIF R² = PEF GAR SPX MXF LVX FIN MAR SAR GMF ENX LOM CIP NOR MIC change [NorA+/-] (fold dilutions) Similar recognition for non phylogenitically-related transporters Vallet et al. ECCMID University of Notre-Dame, Notre Dame, IN 21

22 Substrate specificity of efflux pumps 14 fluoroquinolones; Gram + versus Gram - MIC change [NorA+/-] (fold dilutions) P. aeruginosa vs S. aureus P. aeruginosa vs S. pneumoniae R² = GMF SAR NOR CIP ENX LOM LVX MAR PEF MXF GAR DIF FIN SPX MIC change [PatA+/-] (fold dilutions) R² = SAR GMF NOR CIP MAR ENX LOM LVX FIN PEF GAR DIF MXF SPX MIC change [Mex+/-] (fold dilutions) MIC change [Mex+/-] (fold dilutions) All fluoroquinolones are substrates for broad spectrum transporters from Gram - Vallet et al. (2011) ECCMID University of Notre-Dame, Notre Dame, IN 22

23 Substrate specificity of efflux pumps 24 fluoroquinolones; Gram + (NorA) versus eucaryotic transporter (Mrp4) Accumulation change [Mrp4+/-] (fold) R² = MXF CIP MIC change [NorA+/-] (fold dilutions) Principal component analysis of the correlations between biophysical properties of fluoroquinolones and susceptibility to efflux Correlation between FQ transport by eukaryotic and procaryotic transporters No simple correlation between recognition by transporters and physicochemical properties Dupont et al. (2012) ECCMID University of Notre-Dame, Notre Dame, IN 23

24 Cooperation between prokaryotic and eucaryotic transporters to reduce FQ acativity against intracellular bacteria MIC of Listeria strains and effect of reserpine MIC (mg/l) quinolone EGD CLIP Res. (-) Res. (+) Res. (-) Res. (+) CIP MXF Lismond et al., AAC (2008) 52: University of Notre-Dame, Notre Dame, IN 24

25 Cooperation between prokaryotic and eucaryotic transporters to reduce FQ acativity against intracellular bacteria Wild-type cells and bacteria bacteria overproducing efflux pumps for ciprofloxacin Bacterial efflux is expressed intracellularly Lismond et al., AAC (2008) 52: University of Notre-Dame, Notre Dame, IN 25

26 Cooperation between prokaryotic and eucaryotic transporters to reduce FQ acativity against intracellular bacteria bacteria overproducing efflux pumps for ciprofloxacin Bacteria AND cells overproducing efflux pumps for ciprofloxacin Bacterial and eukaryotic efflux cooperate to reduce ciprofloxacin intracellularly activity Lismond et al., AAC (2008) 52: University of Notre-Dame, Notre Dame, IN 26

27 Cooperation between prokaryotic and eucaryotic transporters to reduce FQ acativity against intracellular bacteria bacteria overproducing efflux pumps for ciprofloxacin Bacteria AND cells overproducing efflux pumps for ciprofloxacin Bacterial and eukaryotic efflux do not affect the activity of moxifloxacin Lismond et al., AAC (2008) 52: University of Notre-Dame, Notre Dame, IN 27

28 University of Notre-Dame, Notre Dame, IN 28 Efflux: from molecular recognition to cellular impact selection of molecules that are poor substrates for efflux may prove useful Molecular determinants for recognition by efflux pumps need to be identified to design poor substrates

29 University of Notre-Dame, Notre Dame, IN 29 Role of antibiotic efflux in epidemiology and resistance.

30 Does efflux mean «resistance» vs. epidemiological breakpoints? MICs vs breakpoints for 183 S. pneumoniae isolated from CAP Efflux (+) strains considered as susceptible FQ with high intrinsic activity can be substrates for efflux! Lismond et al., JAC (2011) 66: University of Notre-Dame, Notre Dame, IN 30

31 Does efflux mean «resistance» vs. epidemiological breakpoints? MICs vs EUCAST breakpoints for 109 P. aeruginosa without or with efflux mechanisms, isolated from ICU patients (VAP) MIC amikacin mexx < 5 mexx 5 mexx expression level MexX substr meropenem mexa < 2 mexa 2 mexa expression level MexA substr pip-tazo MexA substr. mexa < 2 mexa 2 mexa expression level Riou et al, ECCMID University of Notre-Dame, Notre Dame, IN 31

32 University of Notre-Dame, Notre Dame, IN 32 Role of antibiotic efflux in epidemiology and resistance. Efflux confers globally low level of resistance, but it can bring MICs above susceptibility Bkpts Molecules with high intrinsic activity (low MIC) can be excellent substrates for efflux with a risk of seeing their MICs increasing rapidly

33 University of Notre-Dame, Notre Dame, IN 33 Role of antibiotic efflux in intrinsic resistance.

34 University of Notre-Dame, Notre Dame, IN 34 Intrinsic resistance of Pseudomonas to temocillin Temocillin [6- - methoxy-ticarcillin], a carbapenem-sparing drug. -O O S H N OCH 3 S O N O - O O stable to -lactamases, including ESBL Treatment of infections by Gram(-) BUT Pseudomonas is intrinsically resistant to temocillin.

35 Intrinsic resistance of Pseudomonas to temocillin MexAB-OprM mutants are highly susceptible! Efflux responsible for intrinsic resistance Buyck et al, J. Antimicrob. Chemother. (2012) 67(3): University of Notre-Dame, Notre Dame, IN 35

36 Intrinsic resistance of Pseudomonas to temocillin But temocillin is used successfully in Cystic Fibrosis patients Natural mutations in MexAB-OprM restore temocillin activity Buyck et al, J. Antimicrob. Chemother. (2012) 67(3): University of Notre-Dame, Notre Dame, IN 36

37 Intrinsic resistance of Pseudomonas to temocillin Is this clinically relevant? 1/4 of CF strains susceptible! Chalhoub, unpublished University of Notre-Dame, Notre Dame, IN 37

38 University of Notre-Dame, Notre Dame, IN 38 Intrinsic resistance of Pseudomonas to macrolides Azithromycin is widely and successfully used in Cystic Fibrosis patients BUT Pseudomonas is intrinsically resistant.

39 Intrinsic resistance of Pseudomonas to macrolides Azithromycin is widely and successfully used in Cystic Fibrosis patients Kanoh & Rubin; Clin. Microbiol. Rev. (2010) 23: University of Notre-Dame, Notre Dame, IN 39

40 University of Notre-Dame, Notre Dame, IN 40 Intrinsic resistance of Pseudomonas to macrolides Is Pseudomonas «intrinsically» resistant to macrolides? 100 MHB-OprM(-) MHB-OprM(+) cumulative percentage Major role of constitutively-expressed transporters! MIC (mg/l) Buyck et al. Clin Infect Dis. 2012; 55:534-42

41 An intriguing observation MIC (mg/l) Antibiotic Aminoglycosides CA-MHB ph 7.4 ph 5.5 RPMI-1640 Gentamicin Amikacin Tobramycin lactams Piperacillin/Tazobactam Cefepime Ceftazidime Aztreonam Meropenem Fluoroquinolones Ciprofloxacin Polymyxins Colistin Azithromycin 128 > Macrolides regain activity against P. aeruginosa in «eukaryotic» media Buyck et al. Clin Infect Dis. 2012; 55: University of Notre-Dame, Notre Dame, IN 41

42 Why do macrolides express their activity in «eukaryotic» media? ML ML OprM X 1 5 ML ML MexAB-OprM MexXY-OprM prot ML X oprm X prot Buyck et al. Clin Infect Dis. 2012; 55: MHB high MIC RPMI low MIC University of Notre-Dame, Notre Dame, IN 42

43 Why do macrolides express their activity in «eukaryotic» media? University of Notre-Dame, Notre Dame, IN 43 Outer Membrane permeability (%) A MHB NPN B BAL C RPMI ML nitrocefin a MHB b RPMI ML prot 1 ML ML OprM prot ML MexAB-OprM MexXY-OprM oprm MHB high MIC RPMI low MIC

44 Why do macrolides express their activity in «eukaryotic» media? University of Notre-Dame, Notre Dame, IN 44 ML ML OprM 1 ML prot 2 ML prot ML MexAB-OprM MexXY-OprM oprm C 14 -CLR accumulation (ng/mg of prot.) MHB RPMI * MHB high MIC RPMI low MIC Time (h)

45 Why do macrolides express their activity in «eukaryotic» media? University of Notre-Dame, Notre Dame, IN 45 ML ML OprM 1 ML prot 2 ML prot RPMI-1640 CA MHB 3 X oprm relative expression no AZM AZM 1 mg/l * * ML MexAB-OprM MexXY-OprM oprm time (h) MHB high MIC RPMI low MIC

46 Why do macrolides express their activity in «eukaryotic» media? University of Notre-Dame, Notre Dame, IN 46 ML ML 1 X OprM ML ML MexAB-OprM MexXY-OprM MHB high MIC prot 3 ML X oprm X RPMI low MIC prot H-Leucin incorporation (% control) ** MIC *** *** CA-MHB RPMI MIC Azithromycin concentration (mg/l)

47 Why do macrolides express their activity in «eukaryotic» media? University of Notre-Dame, Notre Dame, IN 47 OprM 1 X 5 ML ML MexAB-OprM MexXY-OprM prot ML X oprm X prot MHB high MIC RPMI low MIC

48 Intrinsic resistance of Pseudomonas to macrolides Is this medium effect clinically relevant? CF strains = 345 pneumonia strains = 48 cumulative percentage pneumonia strains 4 MHB RPMI MIC (mg/l) cystic fibrosis strains MHB RPMI > Mustafa, unpublished University of Notre-Dame, Notre Dame, IN 48

49 University of Notre-Dame, Notre Dame, IN 49 Role of antibiotic efflux in intrinsic resistance. Inactivating efflux may reveal antibiotic activity and could be a useful tool when developing new drugs Bacterial responsiveness to antibiotics may be highly different in the host than in the test tube

50 Role of efflux pumps in the clinics University of Notre-Dame, Notre Dame, IN 50

51 Efflux in S. pneumoniae: is it important in the clinics? Suspected efflux based on phenotypic analysis (CIP MIC +/- reserpine) reserpine effect on MIC (x dilutions) % strains acute pathology «one shot» antibiotic exposure 20 0 CAP origin of isolates BPCO 183 strains 107 strains chronic pathology repetitive antibiotic exposures Lismond & Degives, unpublished University of Notre-Dame, Notre Dame, IN 51

52 Efflux in S. pneumoniae: is it important in the clinics? Identification of FQ transporters in clinical isolates Inactivation of pata or patb as efficient as reserpine to reduce MIC responsible for FQ efflux in clinical isolates work as heterodimers Lismond et al, ECCMID University of Notre-Dame, Notre Dame, IN 52

53 University of Notre-Dame, Notre Dame, IN 53 Efflux in S. pneumoniae: is it important in the clinics? SubMICs concentrations of fluoroquinolones may induce efflux systems Optimal dosing is needed! El Garch et al., JAC (2010) 65:

54 Efflux in P. aeruginosa: is it important in the clinics? Prevalence of MexA and MexX overexpressers in 62 phylogentically-related pairs of P. aeruginosa isolated from ICU patients (VAP) DAY 0 (%) DAY x (%) 9.68% 17.74% 11.29% MexA-/MexX- MexA+/MexX % 12.90% 66.13% MexX+/MexA- MexA+/MexX % 22.58% Riou et al, ECCMID University of Notre-Dame, Notre Dame, IN 54

55 Efflux selection in P. aeruginosa during treatment Antipseudomonal antibiotics received by the patients during treatment global influence of treatment Antibiotic no. patients Piperacillin-tazobactam (TZP) 26 Amikacin (AMK) 22 Meropenem (MEM) 20 Cefepime (CEF) 19 Ciprofloxacin (CIP) 6 69% combinations Antibiotic treatment selects for efflux-mediated resistance! number of strains DAY 0 nb efflux systems DAY X number of efflux systems detected at day 0 and day X Riou et al, ECCMID University of Notre-Dame, Notre Dame, IN 55

56 MIC during treatment Increases in MICs of antibiotics used in empirical antipseudomonal therapy between D0 and DX of treatment and this is associated with increase in MICs! Riou et al, IJAA (2010) 36: University of Notre-Dame, Notre Dame, IN 56

57 Early diagnosis should be implemented in the clinics University of Notre-Dame, Notre Dame, IN 57

58 Role of efflux pumps in the clinics University of Notre-Dame, Notre Dame, IN 58 Efflux is present in clinical isolates and may be induced upon exposure to suboptimal antibiotic concentrations Detecting efflux in clinical isolates may help optimizing antibiotic selection and dosing

59 University of Notre-Dame, Notre Dame, IN 59 Antibiotic resistance by efflux: from molecular aspects to cinical impact Take home message My molecular point of view: Appropriate in vitro models may help you selecting antibiotics that are not affected by efflux My clinical point of view: Early diagnosis of resistance mechanisms and optimal dosing are keys to success

60 Efflux and bacteria in our team GRAM(-) L. Avrain M. Riou J. Buyck H. Chalhoub H. Mustafa V. Mohymont GRAM(+) F. el Garch C. Vallet A. Lismond F. Degives N. Vandevelde University of Notre-Dame, Notre Dame, IN 60