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Antimicrobial Chemotherapy and Antibiotic Sensitivity Testing



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Antimicrobial Chemotherapy and Antibiotic Sensitivity Testing

  1. 1. Antimicrobial Chemotherapy and Antibiotic Sensitivity TestingDr.T.V.Rao MD 11/1/2014 Dr.T.V.Rao MD 1
  2. 2. Uses of Antimicrobial Agents •Antimicrobial agents are widely employed to cure bacterial diseases •Definition of Antibiotic –Antibiotics are substances that are derived from a various species of microorganisms and are capable of inhibiting the growth of other microorganism even in small concentrations. 11/1/2014 Dr.T.V.Rao MD 2
  3. 3. Beginning of Antibiotics with Discovery of Pencillin •The discovery of penicillinhas been attributed to Scottish scientist Alexander Flemingin 1928 and the development of penicillin for use as a medicine is attributed to the Australian Nobel Laureate Howard Walter Florey. 11/1/2014 Dr.T.V.Rao MD 3
  4. 4. Discovery of Pencillin 11/1/2014 Dr.T.V.Rao MD 4
  5. 5. Discovery of Pencillin Awarded Nobel Prize 11/1/2014 Dr.T.V.Rao MD 5
  6. 6. Chemotherapeutic Agents •Antimicrobial agents –that are produced synthetically but have action similar to that of antibiotics and are defined as chemotherapeutic agents •Eg Sulphonamides, Quinolones. 11/1/2014 Dr.T.V.Rao MD 6
  7. 7. Definition •Bacteriostatic-Antimicrobial agents that reversibly inhibit growth of bacteria are called as bacteriostic ( Tetracyclnes, Chloramphenicol ) •Bactericical –Those with an irreversible lethal action on bacteria are known as bactericidal ( Pencillin, Isoniazid ) 11/1/2014 Dr.T.V.Rao MD 7
  8. 8. How Drugs Act •Drugs differ on their capabilities to act at different sites on bacteria. •Some drugs have more than one site of action 11/1/2014 Dr.T.V.Rao MD 8
  9. 9. Major mechanisms of antimicrobial drugs •1 Inhibition of cell wall synthesis •2 Inhibition of cell membrane function •3 Inhibition of protein synthesis ( inhibition of translation and transcription of genetic material) •4 Inhibition of nucleic acid synthesis. 11/1/2014 Dr.T.V.Rao MD 9
  10. 10. Pencillin •Pencillin and cephalosporins act inhibiting Trans peptidases, the enzyme catalyses the final linking step in synthesis of peptidoglycan. •Due to this reason Pencillin in bactericidal for grwoing bacteria since new peptidoglycan is synthesized at that stage only. •In nongrwoing cells pencillin is inactive •An intact beta –lactum is essential for antibacterial activity of pencillins 11/1/2014 Dr.T.V.Rao MD 10
  11. 11. Classification of Pencillins •Natural Benzyl pencillin Phenoxymethyl pencillin Pencillin v Semi synthetic and pencillase resistant 1 Methicillin 2 Nafcillin 3 Cloxacillin 4 Oxacillin 5 Floxacillin 11/1/2014 Dr.T.V.Rao MD 11
  12. 12. Extended spectrum pencillins Aminopencillins -Ampicillin, Amoxycillin Carboxypencillins –Carbencillin, Ticarcillin Ureidopencillin -Piperacillin Resistance to pencillin is due to pencillinase commonly called as ßlactamase The enzyme opens Betalactam ring hydrolytically and thus converts the antibiotic to inactive pencillonic acid. 11/1/2014 Dr.T.V.Rao MD 12
  13. 13. Inhibitors to Beta lactamase •Clavulinic acid which is a product of Strept.clavuligerus •Acts against the Staphylococcal beta ßlactamase. •And plasmid mediated Betalactamase of Gram negative bacteria. •Salbactum –this is a semisyntetic sulfone derivative with weak antibacterial activity 11/1/2014 Dr.T.V.Rao MD 13
  14. 14. •Tazobactam –A penicillonic acid sulfone derivative acts well when combined with piperacillin. 11/1/2014 Dr.T.V.Rao MD 14
  15. 15. Cephalosporins •Like pencillin acts similar •Products of the molds of genus Cephalosporium except cefoxilin •Divided into 4 generation of cephalosporins depending on the spectrum of activity. 11/1/2014 Dr.T.V.Rao MD 15
  16. 16. Different Generations of Cephalosporins •Cephalosporins are grouped into "generations" based on their spectrum of antimicrobial activity. The first cephalosporins were designated first generation while later, more extended spectrum cephalosporins were classified as second generation cephalosporins. 11/1/2014 Dr.T.V.Rao MD 16
  17. 17. Major generations of Cephalosporins •Cephalosporins are divided into 3 generations: •1st generation: Cephelexin, cefadroxil, cephradine •2nd generation: Cefuroxime, cefaclor •3rd generation: cefotaxime, Ceftazidime, cefixime -these give the best CNS penetration •4thgeneration Cephalosporins are already available 11/1/2014 Dr.T.V.Rao MD 17
  18. 18. Basis of generations in Cephalosporins •Cephalosporins are grouped into "generations" based on their spectrum of antimicrobial activity. The first cephalosporins were designated first generation while later, more extended spectrum cephalosporins were classified as second generation cephalosporins. 11/1/2014 Dr.T.V.Rao MD 18
  19. 19. Advantages with Newer generations •Each newer generation of cephalosporins has significantly greater gram-negative antimicrobial properties than the preceding generation, in most cases with decreased activity against gram-positive organisms. Fourth generation cephalosporins, however, have true broad spectrum activity 11/1/2014 Dr.T.V.Rao MD 19
  20. 20. Other Beta-lactams include •Other beta-lactams include: •Aztreonam: a monocytic beta-lactam, with an antibacterial spectrum which is active only against Gram negative aerobes, including Pseudomonas aeruginosa, Neisseria meningitidisand N. gonorrhoea. 11/1/2014 Dr.T.V.Rao MD 20
  21. 21. Other drugs •Imipenem: a carbapenem with a broader spectrum of activity against Gram positive and negative aerobes and anaerobes. Needs to be given with cilastatin to prevent inactivation by the kidney. 11/1/2014 Dr.T.V.Rao MD 21
  22. 22. Quinolones •Quinolones are the first wholly synthetic antimicrobials. The commonly used Quinolones. •Act on the DNA gyrase which prevents DNA polymerase from proceeding at the replication fork and consequently stopping synthesis. 11/1/2014 Dr.T.V.Rao MD 22
  23. 23. Aminoglycosides •Aminoglycosides are group of antibiotics in which amino sugars liked by glycoside bonds •Eg Streptomycin, •Act at the level of Ribosome's and inhibits protein synthesis •Other Aminoglycosides – Gentamycin, neomycins,paromomycins,tobramycins Kanamycins and spectinomycins 11/1/2014 Dr.T.V.Rao MD 23
  24. 24. Tetracycline's •Broad spectrum antibiotic produced by Streptomyces species •1. Oxytetracycle, chlortetracycle and tetracycline •Tetracyclnes are bacteriostatic drugs inhibits rapidly multiplying organisms •Resistance develops slowly and attributed to alterations in cell membrane permeability to enzymatic inactivation of the drug 11/1/2014 Dr.T.V.Rao MD 24
  25. 25. Choramphenicol •Choramphenicol is bacteriostatic drug •Can produce bone marrow depression •Chloramphenicol interferes with protein synthesis. 11/1/2014 Dr.T.V.Rao MD 25
  26. 26. Macrolides,Azalides,Ketolides •Contain macro cyclic lactone ring Erythromycin. Is popularly used drug •Other drugs Roxithromycin,Azithromycin •Inhibits the protein synthesis. •Used as alternative to pencillin allergy patients. 11/1/2014 Dr.T.V.Rao MD 26
  27. 27. Other Antimicrobial agents •Lincomycins Clindamycin resembles Macrolides in biting site and antimicrobial activity. Streptogramins Quinpristin / dalfopristin useful in gram postive bacteria 11/1/2014 Dr.T.V.Rao MD 27
  28. 28. Antibiotics in Anaerobes •Major anaerobes –Anaerobic cocci, clostridia and Bacteroides are susceptible to Benzyl pencillin •Bact.fragilis as well as many other anaerobes are treatable with Erythromycin,Lincomycin, tetracycline and Chloramphenicol •Clindamycin is effective against many strains of Bacteroides 11/1/2014 Dr.T.V.Rao MD 28
  29. 29. Metronidazole in Anaerobic Infections •Since the discovery of Metronidazole in 1973 since then it was identified as leading agent anaerobes. •But also useful in treating parasitic infections Trichomonas, Amoebiasis and other protozoan infections. 11/1/2014 Dr.T.V.Rao MD 29
  30. 30. Antiviral drugs •Acyclovir •Ribavirin •Azidothymidine •Amantidine •Idoxuridine 11/1/2014 Dr.T.V.Rao MD 30
  31. 31. Antifungal Agents •Grisofulvin •Imidazole •Polyenes Nystatin, Amphotericin B 11/1/2014 Dr.T.V.Rao MD 31
  32. 32. Antibiotic resistance •Antibiotic resistanceis the ability of a micro organism to withstand the effects of antibiotics. It is a specific type of drug resistance. Antibiotic resistance evolves naturally via natural selectionacting upon random mutation, but it can also be engineered by applying an evolutionary stress on a population. Once such a geneis generated, bacteria can then transfer the genetic information in a horizontal fashion (between individuals) by plasmidexchange. 11/1/2014 Dr.T.V.Rao MD 32
  33. 33. Detection of Antibiotic sensitivity and Resistance 11/1/2014 Dr.T.V.Rao MD 33
  34. 34. Routine Susceptibility Tests •Disk diffusion (Kirby Bauer) •Broth micro- dilution MIC –NCCLS reference method •Etest 11/1/2014 Dr.T.V.Rao MD 34
  35. 35. •Diffusion method –Put a filter disc, or a porous cup/a bottomless cylinder containing measured quantity of drugs on the a solid medium that has been seeded with test bacteria •Dilution method –vary amount of antimicrobial substances incorporated into liquid or solidmedia –followed by inoculation of test bacteria Dr.T.V.Rao MD 35 Antimicrobial Susceptibility Testing 11/1/2014
  36. 36. Susceptibility Testing MethodsInnoculateMH platePlace diskson agar plateIncubate plate18-24 hr, 35 CMeasure and record zone of inhibition around each disk 11/1/2014 Dr.T.V.Rao MD 36
  37. 37. Antimicrobial susceptibility tests Minimum inhibitory concentration [MIC] –The smallest concentration of antibiotic that inhibits the growth of organism Liquid media (dilution) allows MIC estimation Solid media (diffusion) –Disk diffusion (Kirby-Bauer) –E-tests –Allows MIC estimation Beta lactamase production: quick screening method 11/1/2014 Dr.T.V.Rao MD 37
  38. 38. Disk Diffusion Susceptibility Testing Improper agar & disk placement Mueller Hinton agar & good disk placement Use Mueller Hinton agar 11/1/2014 Dr.T.V.Rao MD 38
  39. 39. Antimicrobial susceptibilitytesting using micro-broth dilutions • • • • • • • • • • • • • 96 well microtiter plateug/ml64 32 16 8 4 2 11/1/2014 Dr.T.V.Rao MD 39
  40. 40. Dilution in liquid broth •Tubes containing increasing antibiotic concentrations •Incubation during 18 hr at 37°C •Tedious 0 (Control) 0,25 0,50 1 2 4 8mg/l MIC Bacterial growth Inhibition 11/1/2014 Dr.T.V.Rao MD 40
  41. 41. Kirby-Bauer disc testing Antibiotic-impregnated discs placed on an agar plate at the interface between test organism and susceptible control organism Resulting zones of inhibition compared, use of controls Susceptibility is inferred (standard tables) 11/1/2014 Dr.T.V.Rao MD 41
  42. 42. Antimicrobial Gradient TestingE-test® Read platesafter recommendedIncubationRead MICwhere elipseintersectsscale 11/1/2014 Dr.T.V.Rao MD 42
  43. 43. Dr.T.V.Rao MD 43 E test –MIC Reports are helpful in Critical management decisions •Quantitative MIC data is a prerequisite for the management of critical infections, including sepsis, especially among critical care patients. Etestis 11/1/2014
  44. 44. E-test Plastic strips with a predefined gradient of – One antibiotic – One antifungal Only one manufacturer One strip per antibiotic Wide range of antibiotics Easy to use Storage at -20°C Short shelf life, expensive
  45. 45. Reading E-tests Susceptible <1 Resistant >4 ug/ml Ciprofloxacin for Yersinia pestis Intermediate 1-4 ug/ml Upper reading 11/1/2014 Dr.T.V.Rao MD 45
  46. 46. Are we overusing Antibiotics 11/1/2014 Dr.T.V.Rao MD 46
  47. 47. Choose the Appropriate Antibiotic Think before prescribing Are we using Right drug for the Right bug ? 11/1/2014 Dr.T.V.Rao MD 47
  48. 48. •Program created by Dr.T.V.Rao MD for Medical and Paramedical Students in the Developing world •Email. • 11/1/2014 Dr.T.V.Rao MD 48