1. The Control of Bacterial Growth
ANTIBIOTICS
&
The Control of Bacterial Growth

2. Counting Chamber
Makes thin microscopic preparation in a known volume of liquid

3. Plate Counts
(keep track of the math)

4. Plate counts, continued

5. Plate counts, continued

6. Control of Bacterial Growth
Natural control of bacterial growth

7. Control of Bacterial Growth
Natural control of bacterial growth
Commercial control of bacterial growth
Nitrates, sulfur dioxide
Temperature (eg., Pasteurization, dry heat)
Salt, vinegar

8. Control of Bacterial Growth
Natural control of bacterial growth
Commercial control of bacterial growth
Physical methods of disinfection
Heat
Filtration
Radiation

9. Control of Bacterial Growth
Natural control of bacterial growth
Commercial control of bacterial growth
Physical methods of disinfection
Chemical methods of disinfection

10. Sites of action of germicidal chemicals
Sterilization: removal or destruction of all microorganisms on or in a product
Disinfection: elimination of most or all pathogens on or in a material
Decontamination: reducing pathogens to levels which are safe to handle.

11. The story of Penicillin
Late 1920s
Alexander Fleming
Scottish physician and bacteriologist
Discovered that a fungal metabolite could be used to control bacterial growth
Fungus: Penicillium notatum
Penicillin prevented growth of Staphylococcus aureus

12. The story of Penicillin
"the greatest contribution medical science ever made to humanity."
Time magazine (1999)
The Great Minds Of The Century
“A spore that drifted into his lab and took root on a culture dish started a chain of events that altered forever the treatment of bacterial infections.”

13. Action of penicillin Overlay plate
A colony of the fungus Penicillium notatum is allowed to grow on agar. The plate is overlaid with a thin film of molten agar containing bacteria.
Penicillin production by the fungus creates a zone of growth inhibition of the bacteria.
Penicillin rapidly became the "wonder drug“
One of the single most effective drugs of the last century

14. The extension of human lifespan
Antibiotics
Public health, Sanitation, Immunization

15. Antimicrobial Drugs
Antimicrobial drugs: Low-molecular weight substances, natural or synthetic, which kill or inhibit the growth of microorganisms with relatively little harm to the host.
Antibiotic: a compound naturally produced by molds or bacteria that kills or inhibits the growth of other microorganisms.
Antiviral: a drug that interferes with the infection cycle of a virus.
Bactericidal: kills bacteria.
Bacteriostatic: inhibits the growth of bacteria.

16. Control of Bacterial Growth
Natural control of bacterial growth
Commercial control of bacterial growth
Physical methods of disinfection
Chemical methods of disinfection
Features of antimicrobial drugs
Selective toxicity
Stability
Access to targets

17. Effectiveness of individual antibiotics
Varies with:
the location of the infection
the ability of the antibiotic to reach the site of infection
sensitivity of the bacterial target
Speed of action
Side effect on the host
the ability of the bacteria to resist or inactivate the antibiotic
Access to the world-wide population:
- should be inexpensive and easy to produce and administer
- should be chemically-stable (have a long shelf-life)

18. Location of the infection
Examples:
Certain antibiotics including some of the -lactams do not function very well in the acidic environment of the stomach
Some antibiotics such as bacitracin may be too toxic for internal use but can be used in topical creams for preventing wound infections

19. Ability of the antibiotic to reach the site of infection
Oral antibiotics - simplest approach when effective
Intravenous antibiotics - reserved for more serious cases
Examples:
Antibiotics vary in their ability to:
be absorbed orally
cross the blood brain barrier (BBB)
Vancomycin cannot cross the intestinal lining; administer intravenously
To overcome the BBB: inject large quantities of an antibiotic directly into a patient's bloodstream

20. Sensitivity of target Broad-spectrum
(kill or inhibit a wide range of Gram-positive and Gram-negative bacteria)
versus
Narrow-spectrum
(effective mainly against Gram-positive or Gram-negative bacteria)
Limited spectrum
(effective against a specific species)

21. Speed of action Generally faster:
Bactericidal – drug kills bacteria (penicillin)
Generally slower:
Bacteriostatic – drug inhibits growth (tetracycline)

22. Side effects Antimicrobial therapy:
Optimize the therapeutic index (ratio between the effective dose and the toxic dose)
Stomach Pain/diarrhea
Nausea  
Yeast and other fungal infection 
Allergic reactions
Disrupt function of liver, kidneys and other organs

23. Control of Bacterial Growth
Natural control of bacterial growth
Commercial control of bacterial growth
Physical methods of disinfection
Chemical methods of disinfection
Features of antimicrobial drugs
Mechanisms of action of antibacterial drugs

24. Targets of antibiotic action
Drug target is present in bacteria, but absent in host cells (preferred).
Drug targets a step in metabolism that is essential in bacteria but not in host.
Drug target is present in both bacteria and host, but the bacterial target is more sensitive to drug.

25. Targets of antibacterial medications

26. Peptidoglycan

27. Inhibition of peptidoglycan synthesis
Penicillin interferes with peptidoglycan cross-linking through interaction with Penicillin-Binding Proteins (PBPs).

28. The effect of penicillin on a cell

29. Antibacterial medications that interfere with cell wall synthesis

30. The b-lactam rings of penicillin and cephalosporin

31. The penicillin family

32. Vancomycin Glycopeptide antibiotic
Given intravenously – commonly used for nosocomial infections
Vancomycin binds to the D-ala-D-ala terminal peptide on peptidoglycan precursors and prevents chain elongation
- last resort

33. Antibiotics that inhibit prokaryotic protein synthesis
Anti-ribosomal antibiotics
– second largest class of antibiotics
- selective; structural differences between ribosomes of bacteria and host cells
- affect different stages of protein synthesis
Block initiation
Block chain elongation
oxazolidinones

34. Control of Bacterial Growth
Natural control of bacterial growth
Commercial control of bacterial growth
Physical methods of disinfection
Chemical methods of disinfection
Features of antimicrobial drugs
Mechanisms of action of antibacterial drugs
Determining susceptibility to antimicrobial drugs

35. Determining susceptibility to antimicrobial drugs
Minimum inhibitory concentration (MIC)
Minimum bactericidal concentration (MBC)
Dilution assays and disc sensitivity assays

36. Determining the minimum inhibitory concentration (MIC) of an antimicrobial drug

37. The Kirby-Bauer method for determining drug susceptibility
Size of the zone reflects sensitivity to the drug

38. Control of Bacterial Growth
Natural control of bacterial growth
Commercial control of bacterial growth
Physical methods of disinfection
Chemical methods of disinfection
Features of antimicrobial drugs
Mechanisms of action of antibacterial drugs
Determining susceptibility to antimicrobial drugs
Resistance to antimicrobial drugs

39. Antibiotic resistance
Clatworthy et al Nature Chemical Biology, 2007

40. Antibiotic resistance
Resistance to more than 15 drugs
has become one of the world’s most pressing public health problems
70 percent of nosocomial bacteria - resistant to at least one of the drugs used to treat infections
Some organisms - resistant to all approved antibiotics and must be treated with experimental and potentially toxic drugs
Super bug

41. The selective advantage of drug resistance

42. Mechanisms of acquired antimicrobial resistance

43. Resistance to antibiotics
For an antibiotic to exert its effect it must:
be transported to the site of action
associate with bacteria and penetrate their cell envelope
bind to their specific molecular target
Resistance to drug can occur at any step in this process.
Intrinsic resistance
Acquired resistance: resistance that develops through mutation or acquisition of new genes.

44. Mechanisms of resistance
Synthesis of enzymes that break down the drug
Chemical modification of the drug
Prevention of access to the target site by blocking uptake
Prevention of access to the target by increasing efflux of the drug
Increased activity of an alternative pathway
Modification of the target site

45. -lactams and resistance
The principal mechanism of resistance to -lactams (penicillin) – inactivating enzymes called -lactamases. They hydrolyze the -lactam ring.
Gram-positives produce extracellular -lactamases
Gram-negatives make periplasmic -lactamases

46. Vancomycin

47. Resistance to anti-ribosomal antibiotics
Enzymes modify antibiotics
Pumps actively excrete antibiotics
Methylation of ribosomal RNA
Mutations in ribosomal RNA or ribosomal S50 subunit

48. Quinolones (nalidixic acid)
Inhibit the action of topoisomerases. They trap these enzymes in the act of cutting DNA thus promoting the formation and persistence of double strand breaks in the bacterial chromosome.
Resistance I: mutations in the topoisomerases that prevent/reduce
the binding of the drugs.
Resistance II: efflux pumps remove quinolones from the bacterial cells.

49. Rifampicin Binds to the RNA polymerase to prevent transcription
One of the few drugs that can treat tuberculosis
Resistance: point mutations in RNA polymerase

50. Multidrug therapy Advantage:
The chance that a single bacterium becomes resistant to both antibiotics is small
The effectiveness of the drugs together is greater than that of either drug alone (synergism)
Disadvantage:
The action of one drug reduces the
efficiency of the other (antagonism).
Indifference: Alone or in combination
- no better no worse

51. Control of Bacterial Growth
Natural control of bacterial growth
Commercial control of bacterial growth
Physical methods of disinfection
Chemical methods of disinfection
Features of antimicrobial drugs
Mechanisms of action of antibacterial drugs
Determining susceptibility to antimicrobial drugs
Resistance to antimicrobial drugs
New approaches to discovery of antimicrobial drugs

52. Efforts to identify and synthesize new antibiotics
Continue modification of existing antibiotics
Continue screening of soil isolates
Rational drug design – use crystal structure
of target molecule as guide for design and
synthesis of chemicals that bind to and inactivate the target molecule
Combinatorial chemistry – create an array of chemical derivatives and test this library against particular bacterial targets
Screening of organisms from other environments

53. Inhibitor screen
High-throughput screen of ~ 50, ,000 chemical compound libraries

54. New paradigm for antibiotic development
Traditional antibiotics: kill or inhibit growth of bacteria
New way: target virulence properties
Target bacterial proteins that promote infection
- Prevent colonization
- Block the activity of toxins or other virulence factors
Prevent their binding to receptors or intracellular targets
Prevent their secretion

55. Target virulence properties

56. Three petri dishes

57. Inhibitors of folic acid metabolism