1. Antimicrobial agents PHG 423 1

2. 2 Definitions Antibiotic: Chemicals that are produced by microorganisms that have the capacity to inhibit (static) the growth of, or to kill (cidal), bacteria and other microorganisms Antifungal agent: That kills or inhibits the growth of fungi. Antiviral agent: That kills or inhibits the growth of virus. Anti-infective agents: An agent natural or synthetic that inhibits microorganism or other infective organisms. Selective toxicity: The ability of the antibiotic to kill or inhibit the pathogenic microorganism without affecting the patient cell, this can be achieved when the antibiotic has a specific mechanism of action (specific target) not present in humans and it is effective at very low concentration.

3. 3 Minimal inhibitory concentration (MIC): is the lowest concentration of antibiotic producing inhibition of visible growth. Minimum Bactericidal Concentration is the lowest concentration of antibiotic that yield no growth or results in 100 % decline in the numbers of colonies counted in standardized volume that is subculture overnight in an antibiotic free medium.

4. 4 Microbial Origin of Antibiotics

5. 5 Bacteria –Bacillus sp (Gramicidin, Bacitracin, Polymyxins) Fungi – Penicillum sp (Penicillin) –Cephalosporium sp (Cephalosporins) –Fusidium sp (Fusidic acid) Actinomycetes –Streptomyces sp (Amino glycoside, tetracycline, macrolide) –Micromonospora sp. (Gentamycin and netilmicin)

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11. Antibiotic Spectrum of Activity No antibiotic is effective against all microbes

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13. Mechanisms of Antimicrobial Action Bacteria have their own enzymes for –Cell wall formation –Protein synthesis –DNA replication –RNA synthesis –Synthesis of essential metabolites

14. 14 Mode of Action bactericidal = kill bacteriostatic = stop multiplication Bacteriostatic –E.g. chloramphenicol, tetracyclines –Inhibit bacterial cell replication but do not kill the organism Bactericidal –E.g. penicillins, cephalosporins, aminoglycoside –Cause microbial cell death and lysis

15. 15 Types of Antibiotics

16. 16 * 1910 Arsphenamine * 1912 Neosalvarsan * 1928 Penicillium chrysogenum * 1935 Prontosil * 1936 Sulfanilimide * 1939 sulfacetamide * 1940 sulfamethizole * 1942 benzylpenicillin * 1942 sulfadimidine * 1943 sulfamerazine * 1944 streptomycin * 1947 sulfadiazine * 1948 chlortetracycline * 1949 chloramphenicol * 1949 neomycin * 1950 oxytetracycline * 1950 penicillin G procaine * 1952 erythromycin * 1954 benzathine penicillin * 1955 spiramycin * 1955 tetracycline * 1955 thiamphenicol * 1955 vancomycin * 1956 phenoxymethylpenicillin * 1958 colistin * 1958 demeclocycline * 1959 virginiamycin * 1960 methicillin * 1960 metronidazole * 1961 ampicillin * 1961 spectinomycin * 1961 sulfamethoxazole * 1961 trimethoprim * 1962 cloxacillin * 1962 fusidic acid * 1963 fusafungine * 1963 lymecycline * 1964 gentamicin * 1966 doxacycline * 1967 carbenicillin * 1967 rifampicin * 1968 clindamycin * 1970 cefalexin * 1971 cefazolin * 1971 pivampicillin * 1971 tinidazole * 1972 amoxicillin * 1972 cefradine * 1972 minocycline * 1972 pristinamycin * 1973 fosfomycin * 1974 talampicillin * 1975 tobramycin * 1975 bacampicillin * 1975 ticarcillin * 1976 amikacin * 1977 azlocillin * 1977 cefadroxil * 1977 cefamandole * 1977 cefoxitin * 1977 cefuroxime * 1977 mezlocillin * 1977 pivmecillinam * 1979 cefaclor * 1980 cefmetazole * 1980 cefotaxime * 1980 cefsulodin * 1980 piperacillin * 1981 amoxicillin/clavulanic acid * 1981 cefperazone * 1981 cefotiam * 1981 cefsulodin * 1981 latamoxef * 1981 netelmicin * 1982 apalcillin * 1982 ceftriaxone * 1982 micronomicin * 1983 cefmenoxime * 1983 ceftazidime * 1983 ceftiroxime * 1983 norfloxacin * 1984 cefonicid * 1984 cefotetan * 1984 temocillin * 1985 cefpiramide * 1985 imipenem/cilastatin * 1985 ofloxacin * 1986 mupirocin * 1986 aztreonam * 1986 cefoperazone/sulbactam * 1986 ticarcillin/clavulanic acid * 1987 ampicillin/sulbactam * 1987 cefixime * 1987 roxithromycin * 1987 sultamicillin * 1987 ciprofloxacin * 1987 rifaximin * 1988 azithromycin * 1988 flomoxef * 1988 isepamycin * 1988 midecamycin * 1988 rifapentine * 1988 teicoplanin * 1989 cefpodoxime * 1989 enrofloxacin * 1989 lomefloxacin * 1990 arbekacin * 1990 cefozidime * 1990 clarithromycin * 1991 cefdinir * 1992 cefetamet * 1992 cefpirome * 1992 cefprozil * 1992 ceftibufen * 1992 fleroxacin * 1992 loracarbef * 1992 piperacillin/tazobactam * 1992 rufloxacin * 1993 brodimoprim * 1993 dirithromycin * 1993 levofloxacin * 1993 nadifloxacin * 1993 panipenem/betamipron * 1993 sparfloxacin * 1994 cefepime * 1999 quinupristin/dalfopristin * 2000 linezolid * 2001 telithromycin * 2003 daptomycin * 2005 tigecycline

17. 17 Bacterial Response to Therapy Antibiotic misuse –Use of antibiotics to treat infections they are inactive against (viral infection ) – Use of antibiotics in conditions of noninfectious etiology because of misdiagnosis –Inappropriate prophylactic therapy –Misuse of antibiotics by the patients

18. 18 Bacterial Response to Therapy Host determinants –Sensitivity of MO to the drug –The appropriate dosage –Pharmacokinetics –Duration of therapy

19. 19 Mechanism of Action How the antibiotic perform its effect on micro-organisms –Inhibition of cell wall synthesis (β- lactams) –Destruction of cell membrane (Polyene) –Inhibition of protein synthesis (tetracycline) – Inhibition of nucleic acid function (rifampin inhibits RNA polymerase and fluoroquinolones inhibit DNA gyrase)

20. 20 Combination Therapy In most cases, infection caused by a single defined organism is best treated with a single antibiotic. When organism is exposed to two antibiotics to which it is sensitive, the response to the combination will be: –Antagonism –Synergism Antagonism, the combination of two antibiotics may produce less response than one of the drugs alone. Synergism, a combination of two antibiotics may have a greater effect than the sum of two individual drug effect, it is usually defined as a four-fold or greater decrease in MIC of the individual antibiotic.

21. 21 Mechanisms of Synergy The effect of one drug permits increased entry of the second drug to its site of action inside the cell, e.g. Penicillin and amino glycosides, in which penicillin acts as inhibitor of cell wall synthesis leading to increase the entering of the amino glycoside into the bacterial cell One of the drug inhibits the inactivation of the other drug, e.g. clavulanic acid (β-lactamase inhibitor) and penicillins.

22. 22 Mechanisms of Synergy The two drugs inhibit the different steps in the critical metabolic pathway of organism e.g. sulfonamides ( inhibit folic acid synthesis ) and trimethoprim ( inhibits the reduction of folic acid to THFA). Two drugs interact with different viable enzyme systems inside the bacterial cell.

23. 23 Advantages Synergetic effect. Prevention of resistance. Very useful in mixed infection. Disadvantages It exposes the patient to an increased risk of drug toxicity. It increases the therapy cost. It sometimes be less effective than therapy with a single antibiotic.

24. Fleming and Penicillin

25. Microbial Sources of Antibiotics

26. 26 Fermentation Derived from the latin word fervere which means to boil In biotechnology, any process by which MOs are grown in large quantities to produce any type of useful materials Production in huge amount … fermenters

27. 27 Microbial Growth

28. 28 Growth Curve Lag phase –Adaptation –Should be reduced to avoid the wastage of time and to reduce the medium consumption –Reduced by using of previously inoculated cells Log phase –Exponential growth –Growth rate >> death rate –Primary metabolites production (required either for growth or for energy, e.g., acetic acid, ethanol, citric acid)

29. 29 Growth Curve Stationary phase –Reduced level of nutrients, and accumulation of toxic metabolites –Growth rate = death rate –Secondary metabolites production (toxins, alkaloids, antibiotics, steroids), which produced in response to depletion of nutrients Death or decline phase

30. 30 Growth Medium Minerals & Vitamins (plant growth and differentiation) Carbon/energy source (due to lack of photosynthesis) Growth regulators (cell enlargement, division, and differentiation)

31. 31 Minerals

32. 32 Factors affecting the Growth Temperature (Mesophils (39°C), Thermophiles (60°C), Psychrophiles (4°C)) Oxygen (Aerobic, Anerobic, Faculative, Microaerophilic (low oxygen)) pH (Neutrophiles, Acidophiles, Alkalinophiles) Water (Halophiles (survive in NaCl (1-15%)), Osmophiles (survive in high sugar), Xerophiles (survive in very dry environments)) Light Nutrients

33. 33 Commercial Production of Antibiotics The micro-organism should grow well in submerged culture medium (liquid medium) in a special apparatus called fermentor The antibiotic produced should be released in the liquid medium used. –Optimization of fermentation process through: Medium composition (carbon and nitrogen sours, C/N ratio, vitamins, growth hormones and any additive). Optimum condition of fermentation (temp., PH, aeration, stirring, volume and inoculums size).

34. 34 Commercial Production of Antibiotics Uses of beneficial additives to the medium: –Methionine is added to Cephalsporium spp. To increases the production of cephalosporines. –Phenylacetamide to Penicillum spp. For a high production of penicillin G. –In case of production of tetracycline using Sterpt. aureofaciens, addition of mercaptothiazole (inhibits chlorination of tetracycline) to avoid production of chlortetracycline (highly irritant and very difficult to separated from tetracycline) and increase tetracycline production.

35. 35 Commercial Production of Antibiotics Isolation and purification of antibiotics: Most of antibiotics are released into the culture medium except nystatin, amphotericin B and griseofulvin remain inside the microorganism cells they isolated by extraction of microorganism cells. Most of antibiotics are obtained from growth medium by the following –Selective adsorption e.g. charcoal in case of amino glycosides antibiotics. –Selective precipitation e.g. methyl orange in case of polymyxin production. –Selective solvent - solvent extraction.

36. 36 Aerobic, Gram-positive cocci –Staphylococcus aureus –Staphylococcus epidermidis –Streptococcus pneumoniae –Streptococcus agalactiae –Streptococcus pyogenes Anaerobic, Gram-positive rods –Actinomyces sp. –Clostridium botulinum –Clostridium difficile –Clostridium perfringens –Clostridium tetani Clinically Important Bacteria

37. 37 Aerobic, Gram-positive rods –Bacillus anthracis –Bacillus cereus –Bifidobacterium bifidum –Lactobacillus sp. –Listeria monocytogenes –Nocardia sp. –Rhodococcus equi –Erysipelothrix rhusiopathiae –Corynebacterium diptheriae –Propionibacterium acnes Anaerobic, Gram-positive cocci –Peptostreptococcus Aerobic, Gram-negative cocci Neisseria gonorrhoeae Neisseria meningitidis Moraxella catarrhalis

38. 38 –Vibrio vulnificus –Acinetobacter sp. –Flavobacterium sp. –Pseudomonas aeruginosa –Burkholderia cepacia –Burkholderia pseudomallei –Xanthomonas maltophilia –Vibrio vulnificus –Acinetobacter sp. –Flavobacterium sp. –Pseudomonas aeruginosa –Burkholderia cepacia –Burkholderia pseudomallei –Xanthomonas maltophilia

39. 39 Aerobic, Gram-negative rods –Actinobacillus sp. –Acinetobacter baumannii –Bordetella pertussis –Brucella sp. –Campylobacter sp. –Capnocytophaga sp. –Cardiobacterium hominis –Eikenella corrodens –Francisella tularensis –Haemophilus ducreyi

40. 40 –Haemophilus influenzae –Helicobacter pylori –Kingella kingae –Legionella pneumophila –Pasteurella multocida –Klebsiella granulomatis –Citrobacter sp. –Enterobacter sp. –Escherichia coli –Klebsiella pneumoniae –Proteus sp.

41. 41 –Salmonella enteriditis –Salmonella typhi –Shigella sp. –Serratia marcescens –Yersinia enterocolitica –Yersinia pestis –Aeromonas sp. –Plesiomonas shigelloides –Vibrio cholerae –Vibrio parahaemolyticus

42. 42 –Vibrio vulnificus –Acinetobacter sp. –Flavobacterium sp. –Pseudomonas aeruginosa –Burkholderia cepacia –Burkholderia pseudomallei –Xanthomonas maltophilia

43. 43 Gram-negative spiral –Spirillum minus Anaerobic, Gram-negative cocci –Veillonella sp. Anaerobic, Gram-negative rods –Bacteroides fragilis –Bacteroides sp. –Prevotella sp. –Fusobacterium sp.

44. 44 Atypical –Borrelia burgdorferi –Borrelia recurrentis –Bartonella henselae –Chlamydia trachomatis –Chlamydophila pneumoniae –Chlamydophila psittaci –Coxiella burnetii –Ehrlichia chaffeensis –Anaplasma phagocytophilum –Legionella sp. –Leptospira sp.

45. 45 –Mycobacterium bovis –Mycobacterium tuberculosis –Mycobacterium avium, –Mycobacterium intracellulare –Mycobacterium kansasii –Mycobacterium leprae –Mycobacterium marinum –Rickettsia rickettsii –Orientia tsutsugamushi –Treponema pallidum