Antimicrobial agents : Basic Concepts and Principles

1. İ. Çağatay Acuner M.D., Clinical Microbiologist, Associate Professor Department of MicrobiologyFaculty of Medicine, Yeditepe University, Istanbul cagatay.acuner@yeditepe.edu.tr Antimicrobial agents: Basic ConceptsandPrinciples

2. Evolutionary dynamics of pathogenic bacteriaMicrobiome, Resistome, Parvome, Antibiosis, Hormesis YEDİTEPE UNIVERSITY • Bacteriaareevolving since 3.5 billionsyears • serine-BLs2 evolvedbillion years ago; plasmid encoded since millionsof years • Adaptedtohumans as pathogensca. 12000 years ago (Neolithic Revolution; largerpopulations, agriculture, farming) • ≈ 5 x 1030bacteria on the planet • Humans: 1014cells → 10 % eukaryotic (100 trillioncells in humanmicrobiota) • 10-100 billionbacteria/1 g soil → 4000-10000 species → 1% is cultivable • Antibioticresistance is ancient • Wright GD, Poinar H. 2012. Antibiotic resistance is ancient: implications for drug discovery. Trends Microbiol. 20(4):157-9. • Microbiome: full collection of genes of all the microbes in a community • Resistome: full collection of antibioticresistancegenes in a community • Parvome: range of biologicallyactivecompundsbybacteria, yeasts • 10% screened, 1% known • Antibiosis: small molecule interaction betweenmicroorganisms (producer→harmed) • Hormesis: A dose-dependent responsetobioactive compoundsand drugs • (contrasting activities atlowversushigh)

3. Evolutionary dynamics of pathogenic bacteriaMicrobiome, Resistome, Parvome, Antibiosis, Hormesis YEDİTEPE UNIVERSITY • Antibiotics (ARGDs) have multiple ecological functions : • weapon/shield (arms race, biological warfare) • hypermutator perturbagens (SOS-response, ++) • increases HGT (S. aureus 10-20% genome MGEs) • homeostasis • detoxification • regulation of biosynthesis pathways • regulation of growth and survival

4. Evolutionary dynamics of pathogenic bacteriaMicrobiome, Resistome, Parvome, Antibiosis, Hormesis YEDİTEPE UNIVERSITY • ARGDs in human gut microbiome • Functionalmetagenomics: • 78 ARGDs →R to 13 antibiotics (low homology to pathogen ARGDS) • Aerobic culture+ Functional metagenomics: • 115 ARGDs → R to 13 antibiotics (>>high homology to path ARGDs) • Sommer MO, Dantas G, Church GM. 2009. • Functional characterizationof the antibiotic resistance reservoir in the human microflora. Science325: 1128–31. • ARGDs in water environments • > 90% of bacterial strains MDR (20% R to >5 Abs) • 140 mobile ARGDs in wastewater treatment plant • Szczepanowski,R. et al. (2009). Microbiology 155, 2306–2319. • ARGDs in soil • 110 ARGDs → 12 antibiotics (>high homology to path ARGDs) • 54% of ARDGs previously unknown • Forsberg,K.J., et al. The shared antibiotic resistome of soil bacteria and human pathogens. Science 337, 1107–1111.

5. Evolutionary dynamics of pathogenic bacteriaEvolutionary forces, levels, units, components and phenomena YEDİTEPE UNIVERSITY • Evolution is a change in biological diversity • occuring as a result of • a) generation/acquisition • b) spread/dispersal • c) selection/drift • of variation Arrow of time (no time symmetry; irreversible processes)! No information loss! "Ex unibus plurum et ex pluribus unum" UNIFYING OR DIVERSIFYING CHANGE Baquero F. 2011. The 2010 Garrod Lecture: the dimensions of evolution in antibiotic resistance: ex unibus plurum et ex pluribus unum. J Antimicrob Chemother. 66(8):1659-72. What are the biologically meaningful outcome measurables? Richness (R) (genotype or phenotype frequencies in a population) -subOTU level- Abundance (A) (total density of a population) -OTU, subOTU level- Evenness (E) (Richness Abundance Distributions) -subOTU level-

6. Antibiotic resistance crisis YEDİTEPE UNIVERSITY

7. Antibiotic resistance crisis YEDİTEPE UNIVERSITY

8. Antibiotic resistance crisis YEDİTEPE UNIVERSITY

9. Antibiotic resistance crisis YEDİTEPE UNIVERSITY

10. Antibiotic resistance crisis YEDİTEPE UNIVERSITY

11. Antibiotic resistance crisis YEDİTEPE UNIVERSITY

12. Antibiotic resistance crisis YEDİTEPE UNIVERSITY

13. Antibiotic resistance crisis YEDİTEPE UNIVERSITY

14. Antibiotic resistance crisis YEDİTEPE UNIVERSITY

15. Antibiotic resistance crisis YEDİTEPE UNIVERSITY

16. Antibiotic resistance crisis YEDİTEPE UNIVERSITY

17. Antibiotic resistance crisisSpread of antibiotic resistant bacteria Antibiotic Resistotype Space (resistance-phenome; phenotypic-resistome) Probability Distributions YEDİTEPE UNIVERSITY Acuner I.C. 2006. Theoretical framework on the uses of standardised antibiotic phenotype and pattern indexes. ECCMID 16th, Clin Microbiol Infect 12(s4) P1278., Nice.

18. Antibiotic resistance crisisSpread of antibiotic resistant bacteria Antibiotic Resistotype Space (resistance-phenome; phenotypic-resistome) Probability Distributions YEDİTEPE UNIVERSITY

19. Antibiotic resistance crisisSpread of antibiotic resistant bacteria YEDİTEPE UNIVERSITY

20. VORTEXOGRAPH ARPT15 r=1/40 Rs=38/42 a%=5.3% a=5/75 PDR! Acinetobacter baumannii isolates in the 4 months surveillance period between March-June 2005 at OMU-SUVAM Hospital Acuner I.C. Vortexograph: an informative graphic tool to display antimicrobial resistance surveillance data (Unpublished)

21. Hastane Epidemiyolojisi: Trans-disipliner bir bakışla, yeni kavramlar tanımlanabilir “Epidemi, biyolojik çeşitliliğin* azaldığı bir evrim fazıdır!” Fernando Baquero (*Richness=zenginlik; OTU) Awardee for Excellence in Clinical Microbiology and Infectious Diseases Fernando Baquero, Award Lecture 2005, 15th ECCMID, Copenhagen Epidemi, biyolojik çeşitliliğin (zenginliğin) azaldığı bir evrim fazıdır ! Epidemi ve evrim tanımları benzerdir XXXIII. TMK, Bodrum, 2008, Moleküler Epidemiyolojik Belirteçlerin Seçiminde Kullanılan Ölçütler, Acuner İ.Ç.

22. Hastane Epidemiyolojisi: Trans-disipliner bir bakışla, yeni kavramlar tanımlanabilir “Evrimleşme ve epidemi ilişkilidir!” Fernando Baquero Awardee for Excellence in Clinical Microbiology and Infectious Diseases Fernando Baquero, Award Lecture 2005, 15th ECCMID, Copenhagen Evrimleşme çeşitliliği artırırken, epidemi modüler mobil parçacıkları seçer (seleksiyon) Hastane, modüler ve fraktal evrimsel ve epidemik dinamiklerin etkili olduğu hiper-kompleks bir çevredir XXXIII. TMK, Bodrum, 2008, Moleküler Epidemiyolojik Belirteçlerin Seçiminde Kullanılan Ölçütler, Acuner İ.Ç.

23. Evolutionary dynamics of pathogenic bacteria Evolutionary forces, levels, units, components and phenomena YEDİTEPE UNIVERSITY Canalization/ Buffering Multi-level , multi-unit population dynamics Hard/Soft/Mixed selection Gene-loss Genome-reduction Gene-duplication Gene-conversion Gene-inversion Gene flow Hill-Robertson effect (Clonal interference in asexuals) Perturbagen/ Environmental feedback Social traits: Cooperation, Altruism, Selfishness, Spite Kin recognition/ Kin selection Group selection Truncation selection Local fitness peak/ Adaptive valleys/ Travelling waves/ Range expansion Founder effect Purging Extinction Metapopulation dynamics Abiotic/Biotic stochasticity Finite/Infinite Populations Non-/Overlapping generations Directional/Positive selection (dN/dS>1) “Stabilizing/Purifying selection (dN/dS<1) Disruptive/Diversifying selection dN/dS=1 → Neutral evolution Evolution of Evolvability/ Robustness Bounded/Unlimited Growth Effective Population Size (Ne) Hypermutation/ Normomutation/ Hypomutation “Muller’s ratchet”/ Mutational meltdown Genomic/Phenomic Plasticity Genetic “hitch- hiking” Distribution of fitness effects Mutation rate, HGT/LGT rate (r/μ) Fitness Landscapes/ Fitness Flux/ Free Fitness/ Flattest Func Genom Architecture “Frequency-dependent selection” “Density-dependent selection” Selective gradients/compartments Fitness gradient (selection coefficient) "Bet-hedging" (Conservative/Diversified) Life-history Spatial structure/heterogeneity Punctuated/Gradual Evolution Shifting balance Hysteresis Epigenetic errors Macro-/Micro-Evolution (Divergence ↔ Convergence) Random Drift and Fixation Migration/ Dispersal/ Sexual-Reproductive Isolation Modularity Re-arrangement (intragenic recombination) Epistatis and Linkage Disequilibrium Near Neutrality Population bottleneck Selection Types and Strength Types of Molecular Evolution Molecular Clock Genetic Draft Periodic selection, Selective sweep Epistasis (Negative/Positive Sign/Reciprocal sign/ Magnitude/ Diminishing returns/ Higher-order) Quasispecies dynamics Path ensemble (of evolutionary trajectories) Detailed Balance “Chance, Necessity, Oppurtunity”

24. Evolutionary dynamics of pathogenic bacteria Macroevolution Tree of Life versus Net of life/Ring of Life (LGT/HGT Effect) YEDİTEPE UNIVERSITY

25. Evolutionary dynamics of pathogenic bacteria “Host-parasite co-evolution”“Continuum of micro-evolution and macro-evolution”(concepts proposed by May, Anderson, Tibayrenc, Baquero and others) YEDİTEPE UNIVERSITY

26. Evolutionary dynamics of pathogenic bacteria “Host-parasite co-evolution”“Transitivity of epidemiological phenomena and evolution” (concepts proposed by May, Anderson, Tibayrenc, Baquero and others) YEDİTEPE UNIVERSITY

27. list antimicrobial susceptibility testing methods (bacteria) • Antimicrobial Disk Susceptibility Methods (Tests) (EUCAST, CLSI) • Antimicrobial Gradient Strip Susceptibility Tests (Commercial) • Antimicrobial Gradient Plate Susceptibility Method (Commercial) • Antimicrobial Dilution Susceptibility Methods (EUCAST, CLSI) • Agar Dilution (Agar Plate) • Microdilution (Microplate) • Macrodilution (Tube)

28. describe disk diffusion susceptibility testing method (bacteria)Antimicrobial Disk Susceptibility Methods (Tests) (EUCAST, CLSI) • http://www.eucast.org/

29. describe disk diffusion susceptibility testing method (bacteria)Antimicrobial Disk Susceptibility Methods (Tests) (EUCAST, CLSI) • http://www.clsi.org/

30. describe disk diffusion susceptibility testing method (bacteria)Antimicrobial Disk Susceptibility Methods (Tests) (EUCAST, CLSI) • The Kirby-Bauer method is the standard recommended by FDA and CLSI • This method is highly standardized • Mueller-Hinton Agar medium is used. • Petri dishes to a uniform thickness of 4 mm. • Inoculated from a broth culture having a turbidity of 0.5 McFarland unit. • Antibiotic disks containing certain amounts of antibiotics • Incubated for 16-18hrs. • Inhibition zones (mm) measured. • Effectiveness of each antibiotic is defined.

31. describe disk diffusion susceptibility testing method (bacteria)Antimicrobial Disk Susceptibility Methods (Tests) (EUCAST, CLSI)

32. describe disk diffusion susceptibility testing method (bacteria)Antimicrobial Disk Susceptibility Methods (Tests) (EUCAST, CLSI)

33. describe disk diffusion susceptibility testing method (bacteria)Antimicrobial Disk Susceptibility Methods (Tests) (EUCAST, CLSI)

34. describe disk diffusion susceptibility testing method (bacteria)Antimicrobial Disk Susceptibility Methods (Tests) (EUCAST, CLSI)

35. describe disk diffusion susceptibility testing method (bacteria)Antimicrobial Disk Susceptibility Methods (Tests) (EUCAST, CLSI)

36. Antimicrobial Gradient Strip Susceptibility Tests (Commercial)

37. Antimicrobial Gradient Strip Susceptibility Tests (Commercial)

38. Antimicrobial Dilution Susceptibility Methods (EUCAST, CLSI)Microdilution (Microplate)

39. describe disk diffusion susceptibility testing method (bacteria)Antimicrobial Disk Susceptibility Methods (Tests) (EUCAST, CLSI)

40. MIC Value of an Bacterial Isolate from a Patient

41. Pharmacokinetics (PD)Critical Concentration (per DDD=c or per maxD=C)

42. Pharmacodynamics (PD)Time over MIC

43. Pharmacodynamics (PD)Area Under Curve/MIC Inhibition Cooefficient

44. Pharmacodynamics (PD)

45. Pharmacodynamics (PD)

46. Setting Breakpoints for Susceptible/Intermediate/Resistant Categories

47. Setting Breakpoints for Susceptible/Intermediate/Resistant Categories

48. Setting Breakpoints for Susceptible/Intermediate/Resistant Categories

49. Setting Breakpoints for Susceptible/Intermediate/Resistant Categories

50. MDR, XDR, PDR Definitions by ECDC, 2011