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1. Antimicrobial therapyMUDr. Lenka Černohorská, Ph.D. Antibiotics are substances against bacteria Other groups: Antivirotics –against viruses Antituberculotics - against mycobacteria Antiparasitics – against parasites

2. Antibiotics are devideddue to mechanismofeffectinto 4 groups: • Inhibition of cell wall synthesis (betalactames, glycopeptides) • Cell membrane destroy (polypeptides) • Inhibition of NA syntesis (quinolons, imidazols) • Inhibition of proteosyntesis (tetracyclines, chloramphenicol, macrolides, lincosamides, aminoglycosides) • Attack against bacterial metabolism (sulfonamids)

3. Betalactames • Baktericidal, only for growing bacteria • Often causes allergy • Penicillins (PNC, oxacillin, ampicillin, piperacillin) • Cefalosporines (1.- 4. generation) • Monobactams (aztreonam) • Carbapenems (imipenem, meropenem)

4. Glycopeptides • Reservedfor G+ bacteria • Vancomycinandlesstoxic, but more expensiveteicoplanin Polypeptides • Ototoxicandnefrotoxic • Polymyxin Bonlylocal as part ofear drops - Otosporin • Polymyxin E – colistinrareused • Primary resistence: all G+ bacteria, proteus, providencia, morganella, serratiaetc. N

5. Aminoglycosides • Bactericidal,ototoxicity and nefrotoxicity • Synergy with betalactames – decrease of toxicity • Preparates: Streptomycin only against tuberculosis, gentamicin,netilmicin, amikacin,neomycin with bacitracin = framykoin (neomycin is too toxic, only for local using)

6. Tetracyklines • Broad spectrum • Don‘t use until 10 years (teeth development) • Less used Chloramphenicol • Broad spectrum • Good penetration to liquor, Hematotoxicity

7. Macrolides I. generation: erythromycin, rare used II. generation: roxithromycin III. generation: clarithromycin, azithromycin – good intracellular penetration and longlasting effect, for G+ bacteria Lincosamides • Lincomycin and clindamycin • Reserved for surgery, good effect to G+ bacteria and anaerobes in addition to Clostridium difficile – risc of pseudomembranous enterocolitis

8. „ Quinolones • Bactericidal • Don‘t use until 15 years (growth cartilages) • I. generation (oxolin acid), II. generation (norfloxacin) only for urinary infection • III. generation: ofloxacin, ciprofloxacin – also for systemic infection – often used

9. Analogs of folate acid • Sulfametoxazol in combination with trimetoprim form co-trimoxazol known as BISEPTOL • Bacteriostatic, worse penetrate into tissues • Nitrofurantoin (and nifuratel) • Effectivity on sugar metabolism. Bacteriostatic, broad spectrum • For urinary tract infection. Weighty undesirable effect: GIT disorder etc. Other antibiotics Linezolid (zyvoxid) – against resistant staphylococci

10. Nitroimidazols • For anaerobes, for protozoas (T. vaginalis etc.) • Metronidazol, Ornidazol Antituberculotics • HRZS,HRZE - starting therapy (INH, rifampicin, pyrazinamid, streptomycin, etambutol) + other • HRZ,HRE – sequenced therapy

11. Antivirotics • Against herpes – acyclovir… • CMV – gancyklovir, foscarnet • Influenza – amantadin, rimantadin, tamiflu • Antiretrovirus therapy – inhibitors of reverse transcriptase (nucleosid+nonucleosid) , inhibitors of protease – in combination Preparates: zidovudin, didanosin …

12. Antimycotics • Fluconazol, itraconazol, ketoconazol etc. – local (vaginal, skin infection) • Amphotericin B – i.v. (in sepsis) Antiparasitics • Against protozoa, helmintes, ectoparasites (moore in parasite capitol) Other preparates • Antimalaric: primachin, chlorochin, meflochin… • Leprosity: dapson

13. Susceptibility testing in vitro • Therapy does not always correspond with susceptibility testing • Quantitative tests (MIC, E-tests) – in relevant patients • Qualitative tests (disc diffusion method) – enough for common cases (susceptible - resistant)

14. Disc diffusion test • MH agar is inoculated with suspension of bacteria • Antibiotic discs (paper impregnated with antibiotics) are applied at MH – atb diffuse from disc through agar • Concentracion of atb decrease with distance from disc • If microb grow to disc/if there is little zone - is resistant (not susceptible) • Big zone (higher than defined size) means susceptibility.

15. Disc diffusion test + via dispensor aplied 6 papers impregnated with antibiotics Cultivation + (37°C/18-24h) ATB diffuses from paper MH medium inoculated with bacteria R Interpretation: High zone= susceptible bacteria (S) Small/Any zone=resistant bacteria (R) s R

16. Microdilution test (MIC) • MIC is the lowest concentration, which inhibites growth(first clear row) • On paper stencil is asigned breakpoint. If MIC is lower than breakpoint, bacterium is susceptible. If MIC is higher, bacterium will be resistent • 1 plastic plate is used for 1 bacterium, for 12 antibiotics, in 8 various (decreasing) concentracion (12th only in 7, because corner row upper right is growth control)

17. MIC – Material and methods 1 row is a growth control inoculation of plate with pipette cultivation (37°C/18-24h) bacterial suspension (0,5-1 CFU) microtiter plate with 96 rows reading

18. Interpretation Case -susceptible Case - resistant 4

19. PEN AMS CXT CLI CMP MTR PEN AMS CXT CLI CMP MTR 4 64 128 32 64 64 4 64 128 32 64 KR 2 32 64 16 32 32 2 32 64 16 32 32 1 16 32 8 16 16 1 16 32 8 16 16 0.5 8 16 4 8 8 0.5 8 16 4 8 8 0.25 4 8 2 4 4 0.25 4 8 2 4 4 0.125 2 4 1 2 2 0.125 2 4 1 2 2 0.063 1 2 0.5 1 1 0.063 1 2 0.5 1 1 0.031 0.5 1 0.25 0.5 0.5 0.031 0.5 1 0.25 0.5 0.5 Interpretation of MIC - antibiogram – goes to clinician! PEN (penicillin)….4……resistant AMS (unasyn)……2…..susceptible

20. E-tests (quantitative) • Similar to disc diffusion test, but strip is used • An increasing concentration of atb is used. Zone is egg like. • There is a scale on strip – simply reading MIC value is 0,75 mg/l (where borderline of zone cross the scale)

21. Resistance of microbes to antibiotics • Primary resistance: all strains of bacteria are resistent. • Secondary resistance: arises unsensitive mutants, by selective antibiotics pressure became dominant * MBC (minimum bactericidal concentracion) isthe lowestconcentration, whichkills bacteria Primary bactericid: atb, where MIC and MBC are almost equal Primary bacteriostatic: atb, where MBC is X-fold higher than MIC - unreal baktericidal effect in human body

22. Resistance factors detection • Special detection methods for resistance factors (for ex. betalactamase). It can be diagnostic strips (chemical detection of specific ensym) or other tests (ESBL) 1. Betalactamase testing • In neisseria, M. catarrhalis, H. influenzae • destroys betalactams • For therapy we useATB with inhibitors of betalactamase likeclavulanate, sulbactam…

23. Detectionof betalactamase Paper with substrate + moisturingsolution Petri dish with bacteria Touch Reaction end (yellow) Colour change (red) Strain produces betalactamase After 30 sec red is missing

24. 2. ESBL (extended spectrum betalactamase) E. coli, K. pneumoniae etc. produces ESBL, whichdestroyscheap betalactams. For therapy we useexpensive carbapenems, aminoglycosides (toxicity),problem of ICU, big hospitals ESBL – screening • Inhibitionof growth between discs – owing to a synergism of 2-3 antibiotics such as aztreonam, AMC, ceftriaxon aztreonam Amoxicilin/clavulanate

25. ESBL detection (CLSI) • 4 discs: Cefotaxim (1) and ceftazidim (2), cefotaxim with clavulanate (3) and ceftazidim with clavulanate (4) • Difference between cefalosporines (1,2) and cefalosporineswithclavulanate (3,4) is higher than 5mm Compare 1 with 3 and 2 with 4 3 1 4 2

26. ESBL andAmpCdetection set (A,B,C,D) Susceptible strain AmpC+ A B A B A cefpodoxime (CPD)B CPD+ESBL inhibitor C CPD+AmpC inhibitor D CPD+both inhibitors C D C D ESBL+,AMPC+ ESBL+ A B A B C D C D Interpretation: compare inhibition zones, if differences are higher than 5 mm