1. By As. Prof. O.Pokryshko Chair of Medical biology, Microbiology, Virology, and Immunology Antibiotics, classificaions and mechanism of action. The main principles of rational antibiotic therapy of diseases.

2. Lectures schedule 1. History of antibiotics discovery. 2. Classification of antibiotics. 3. Examination of bacterial susceptibility to antibiotics. 4. The main principles of rational antibiotic therapy of diseases. 5. Complication of antibioticotherapy.

3. - Diarrheal diseases - 4 billions cases, - Malaria - 500 mln, - acute infection of respiratory tract - 395 mln, - sexual transmitted diseases - 330 mln, - measles - 42 mln, - whooping cough - 40 mln - tuberculosis – 1,9 bln of infected persons, 9 mln of new cases of diseases - AIDS – 50 mln cases, 6 mln people died - SARS, hemorrhagic fever

4. Modern chemotherapy has been dated to the work of Paul Ehrlich (Germany) Ehrlich postulated that it would be possible to find chemicals that were selectively toxic for parasites but not toxic to humans. He introduced the concept of chemotherapy dealing with the treatment of diseases with chemicals. This idea has been called the "magic bullet" concept.

5. A. Fleming The first antibiotic, Penicillin G, was discovered in 1929 by Alexander Fleming. 1940s: Penicillin was tested clinically and mass produced.

6. He observed that Penicillium fungus made an antibiotic, penicillin, that killed S. aureus. Figure 1.5

7. In 1944 Waksman isolated streptomycin and subsequently found agents such as chloramphenicol, tetracyclines, and erythromycin in soil samples. S. Waksman

8. In 1939 Florey and colleagues at Oxford University again isolated penicillin G. FloreyE. Chainy

9. Antibiotics Antibiotics are chemical substances produced by microorganisms (such as bacteria, fungi, actinomyces) or other organisms which suppress the growth of other microorganisms and eventually destroy them. Some antibiotics have been produced by chemical synthesis or semi- synthetically from natural substances.

10. The term “antibiotics” proposed in 1942 S. Waksman It means: anti – against, bios - life

11. Microbial antagonism is the basis of modern use of antibiotics L. Pasteur

12. Peculiarities of antibiotics - high level of biological activity - high election specificity

13. Activity of antibiotics is evaluated in International Unit

14. Classification of antibiotics according to their origin 1. Antibiotics from fungi:  Penicillins (Penicillium notatum, P.chryzogenum),  Cephalosporins (Cephalosporium salmosynnematum),  Griseofulvinum (P. griseofulvum, P. patulum, P. nigricans),  Fusidin (Fusidium coccineum),

15. 2. Antibiotics from Actinomyces:  Aminoglicosides: Streptomycin (Streptomyces griseus), neomycin (S. fradiae), kanamycin (S. kanamyseticus), tobramycin (S. tenebrarius), gentamycin (Micromonospora purpurea), sisomicin (Micromonospora inyoensis);  Tetracyclines: chlortetracycline (S. aureofaciens), oxytetracycline (S. rimosus);  Chloramphenicol (S. venezuelae);  Macrolides: oleandomycin, (S. antibioticus), erythromycin (S. erythreus),

16. 2. Antibiotics from Actinomyces:  Linkomycin (S. lincolniensis); е) Rifampicin (S. mediterranei);  Polyenes: nystatin (S. noursei), levorin (S. levorys Krass), amphotericin B (S. nodosus);  Inhibitors of beta-lactamases: klavulanic acid (S. clavuligerus),  Carbapenem (S. olivaceus),  Thienamycin (S. cattleya).

17. 3. Antibiotics from bacteria:  Bacillus: polymyxin (B. polymyxa), licheniformin (B. licheniformis), gramicidin С (B. brevis), subtilin (B. subtilis);  Pseudomonas: piocianin (P. aeruginosa), sorbistin (P. sorbistini),  other bacteria: monobactams (Chromobacterium violaceum), nisin (Streptococcus lactis), prodigiosin (Serratia marcescens), coliformin (E. coli), streptosin, diplococcin (Streptococcus spp.), azomycin, nocardamin (Nocardia)

18. 4. Antibiotics from plants  Chorellin (Chlorella vulgaris);  Arenarin (Helichrysum arenarium);  Gordecin (barley);  Chinin (cinchona tree);  Alicin (garlic, Allium sativum);  Raphanin (radish, Raphanus sativum);  Phaseolin (haricot bean, Phaseolus vulgaris).

19. 5. Antibiotics from animal tissues:  interferons (spleen, macrophages, tissue cells),  lysozyme (most body fluid, salive, eggs);  erythrin (red cells, liver);  ecmolin (fish)

20. Classification of antibiotics according to the spectrum of biological action 1. Antibacterial: А. Narrow spectrum of action which are active against gram-positive bacteria: а) natural Penicillins; b) semi- synthetic Penicillins (methicillin, oxacillin); c) Cephalosporins; d) Lincomycin; е) Macrolodes. Б. Broad spectrum of action а) semi-synthetic Penicillins (Ampicillin, Amoxicillin); b) Cephalocporins of ІІ-IV generation; c) Tetracyclines; d) Chloramphenocol; e) Aminoglycosides; f) Polymixins; g) Fluoride quinolones

21. 2. Antifungal (amphotericin). 3. Antiviral (amantadin, vidarabin). 4. Antiprotozoal (emethin, chinin). 5. Antineoplastic (bleomycin, mitomycin C, actinomycines). Classification of antibiotics according to the spectrum of biological action

22. Spectrum of Activity Relates to the number of microbes that are susceptible to the action of the drug –Narrow (limited number) / Broad (wide) Penicillin G is a narrow spectrum drug as it is only effective against gram-positive microbe Tetracyclines are effective against gram-positive and gram-negative microbes (Broad) Note: Never confusion these terms with potency levels of the drugs or efficacy (ie. Narrow are weak, Broad are strong)

23. Bactericidal agents are more effective, but bacteriostatic agents can be extremely beneficial since they permit the normal defenses of the host to destroy the microorganisms. Bacteriostatic  Reversible inhibition of growth  When the antibiotic is removed, almost all of the bacteria can replicateBactericidal  Irreversible inhibition of growth  When the antibiotic is removed, almost none of the bacteria (10-7to 10-3) can replicate Classification of antibiotics according to the spectrum of action

24. Mechanism of Action: 1.Inhibition of Cell Wall Synthesis 2.Disruption of Cell Membrane 3.Inhibition of Protein Synthesis 4.Interference with Metabolic Processes NB: Bactericidal Bacteriostatic

25. Inhibition of Cell Wall Synthesis Most bacteria possess a cell wall to protect from osmotic pressures Microbe divides – needs to create a new cell wall –Interrupt this leads to new microbes being susceptible to external influences –Cell ruptures  Microbe death Eg. Penicillinsm cephalosporins, vancomycin and bacitracin

26. Disruption of the microbial cell membrane Essentially, affect cell membrane transportation in and out Increases permeability of membrane –External influences have greater effect –Microbe death Eg. Polymyxin, Colistin Note: These agents are more toxic systemically than those agents that inhibit cell wall synthesis.

27. Inhibition of Protein Synthesis Proteins vital for growth and repair Act either at: –Site of protein synthesis (ribosome) –Within the nucleus by inhibiting synthesis of nucleic acids DNA replication / RNA synthesis = TRANSCRIPTION Eg. Tetracyclines, aminoglycosides and macrolides (erythromycin) Exploit structural differences between microbial and human cells –High dose can lead to toxicity

28. Interference with metabolic processes Agents are structurally similar to Para- aminobensoic acid (PABA) – component of folic acid –Essential for nucleic acid synthesis, without it microbes can not produce the proteins for growth –Exploits: microbes need to create their own folic acid, whilst we get it in our diets. Eg Sulphonamides, Trimethoprim

30. Examination of susceptibility of bacteria to antibiotics  Serial dilutions: - in a liquid medium - in a solid medium  Disc diffusion method  Rapid methods

31. Demands to nutrient media 1.to be standard and provide optimal conditions for microbial growth; 2.do not have inhibitors of bacterial growth and a lot of stimulators; 3.do not have substances, which inhibit antibiotic activity

32. Disc diffusion method

34. Serial dilution in liquid medium

35. Serial dilution in solid medium

36. Minimal Inhibitory Concentration (MIC) Lowest concentration of antibiotic that prevents visible growth Broth or tube dilution methodSerial 2-fold dilutions of the antibiotic Accurate but time-consuming Disk sensitivity testRapid, but must be related to results from the tube dilution method

37. Minimal Bactericidal Concentration (MBC) Lowest concentration of antibiotic that reduces the number of viable cells by at least 1000-fold Performed in conjunction with MIC by the tube dilution methodAliquots from the tubes at and above the MIC are plated onto agar media The antibiotic is diluted, so that the remaining viable cells grow and form colonies –The MBC of a truly bactericidal agent is equal to or just slightly above its MIC

38. Rapid methods  examination of changes of microbial enzymes activity under the influence of antibiotics;  examination of color of redox- indicators;  cytological evaluation of morphological changes;  automatic

39. Automatic metod of examination of bacterial susceptibility

40. Criteria that determines the effectiveness of antimicrobial agents used in the treatment of infectious diseases: 1. Selective toxicity - destroys or inhibits microbe without affecting host cells 2.Broad spectrum - effective against a wide variety of organisms 3.Non-mutagenic - does not induce development of resistant strains 4.Soluble in body fluids - distributed through body (in bloodstream)

41. Criteria that determines the effectiveness of antimicrobial agents used in the treatment of infectious diseases: 5.Stable in body fluids - not easily broken down or excreted, to maintain constant and effective levels 6.Absorbed by tissues - to reach site of infection 7.Non-allergenic to host - should not cause adverse reactions in host 8.Should not disturb host’s normal flora (organisms normally living in body) causing secondary (super) infections produced by opportunists

42. General principles 1. The first question to ask before prescribing an antibiotic is whether its use is really necessary. There is no point in prescribing it if, for instance, the disease is not due to an infection (fever does not always indicate the presence of an infection), or if the infection is due to agents such as viruses, which do not respond to antibiotics. All therapy is a calculated risk in which the probable benefits must outweigh the draw­backs, and antibiotics are no exception to this rule. To use them when they are not indicated and when the "probable benefits" are non- existent means exposing the patient to the risk of adverse reactions, or worse.

43. 2. Patients with similar infections react differently. This may be due to previous contact with the same pathogen or to the individual immune response. The presence of hepatic or renal disease may necessitate changes in the dosage or the choice of antibiotic. Knowledge of any past adverse reactions to antibiotics is also essential. 3. The doctor must be familiar with the typical response of infections to proper antibiotic treatment. Acute infection with group A streptococci or pneumococci responds rapidly (usually within 48 hours) to penicillin G, while the temperature curve in typhoid fever treated with chloramphenicol may not show any change for four or five days.

44. 4. The doctor must know which bacteria are commonly found in which situations, for instance Pseudomonas in extensive burns (sepsis is frequent and often fatal) and in the expectoration of children with cystic fibrosis, or Streptococcus pneumoniae and Haemophilus influenzae in chronic bronchitis of the adult.

45. 5. Ideally, treatment with antibiotics should not be instituted before samples for sensitivity testing have been collected. Such tests can be dispensed with, however, when the causative organism is known and its response to the antibiotic is predictable. But the sensitivity of, for instance, many gram-negative strains can change, even during treatment, making an alternative treatment necessary. In addition, the clinical results may be at odds with the findings of the sensitivity tests. Even a severe infection may show a satisfactory clinical response despite apparent lack of sensitivity.

46. Antibiotic treatment is considered a failure if no response is seen within three days. Failure may be due to various causes: 1. Wrong diagnosis (a viral infection does not respond to antibiotics). 2. Wrong choice of antibiotic. 3. Wrong dosage (wrongly dosed by doctor or poor patient compliance). 4. Development of resistance during therapy (as sometimes occurs in tuberculosis and infections due to gram-negative pathogens). Failure Failure of antibiotic therapy

47. 5. Superinfection by resistant bacteria. 6. Accumulation of pus necessitating surgical drainage (buttock abscess). 7. Underlying disease (lymphoma, neoplasia) of which the infection is only an intercurrent complication. 8. Drug fever. Failure of antibiotic therapy

48. І. Allergic reactions ІІ. Toxic effects on normal tissues ІІІ. Disturbs host normal flora  secondary infections (Dysbacteriosis) Disadvantages of Antimicrobial Therapy:

49. Disadvantages of Antimicrobial Therapy: of Antimicrobial Therapy: І. Allergic reactions - dangerous for life (anaphylactic shock, angioneurotic oedema of larynx) - non-dangerous for life (skin itching, urticaria, rash, rhinitis, glossitis, conjunctivitis, photodermatoses (tetracyclines)

50. ІІ. Toxic reactions - dangerous for life (agranulocytosis, aplastic anemia, endotoxic shock) - non-dangerous (neuritis of N. vestibularis and N. auricularis - aminoglycosides; periferal neuritis, vomiting, nausea, diarrhea, hepatotoxic and nephrotoxic effects, embriotoxic effect, pigmentation of the teeth) Disadvantages of Antimicrobial Therapy: of Antimicrobial Therapy:

51. ІІІ. Dysbacteriosis - dangerous for life (generalized candidiases sepsis, staphylococcal enterocolitis, secondary pneumonia, which cause gram-negative bacteria) - non-dangerous for life (local candidiases) Secondary action of antibiotics

52.  Natural resistance  Acquired resistanse  primary  secondary Types of resistance

53. Mechanisms of Resistance Development of resistant strains – spontaneous mutations, DNA transfer a.Ability to destroy AMA by producing enzymes (Staph –penicillinase or  -lactamase) b.Mutations causing structural changes in cell so bypass metabolic step inhibited by AMA (L-forms - no cell wall) c.Over produce target molecules  increase in quantity overcomes action of AMA d.R-factors (resistant genes) in plasmids transferred to bacterial cells by conjugation, transformation, transduction

57. R-Plasmids Resistance transfer factors, or RTFs

58. Transposons Staphylococci, Enterobacteria – transposon Tn551 (erythromycin), Tn552 (penicillin), Tn554 (erythromycin, spectinomycin). They can integrate with R-plasmids and phages

59. Mechanism to Reduce Bacterial Resistance. Proper selection of new antibiotics will be a major force in slowing the development of antimicrobial resistance. Proper hygiene practices will reduce plasmid transfer and the establishment of multiple drug-resistant bacteria in the hospital and will delay the appearance of such species in the community. There are a number of mechanisms to prevent bacterial resistance. The health care provider must be continually alert to the appearance of antibiotic resistance within the hospital and community.