2. Chemotherapy of Bacterial Infections ~~~~~~~~ Antibiotics

3. Definitions of Antibiotics
OLD: An antibiotic is a chemical substance produced by various species of microorganisms that is capable in small concentrations of inhibiting the growth of other microorganisms
NEW: An antibiotic is a product produced by a microorganism or a similar substance produced wholly or partially by chemical synthesis, which in low concentrations, inhibits the growth of other microorganisms
What is the role of antibiotic in nature:
Germ Warfare- the production of compounds that discourage microbial competitors. Problem with this explanation is that antibiotic production by microbes growing in nature is so low that levels of antibiotics are undetectable.
May be signaling molecules
NO EVIDENCE FOR EITHER ROLE

4. Impact of Modern Healthcare on Life Expectancy
One of the greatest advances in human health during the past century was the discovery that our natural defenses could be augmented with externally provided defences:
ANTIBIOTICS, DISINFECTANTS AND ANTISEPTICS
Easy to forget how much chemotherapy has impacted on the world. Ask people over 75 about losses due to pneumoniae or post operative infections.
NOTE: recent advances haven’t impacted on lifespans much.

5. History Paul Ehrlich “Magic Bullet” Chemicals with selective toxicity
ORIGIN: Selective Stains
DRUG: Arsphenamine (1910)
“606” Salvarsan
NOBEL: 1908
Paul Ehrlich- derived from finding that dyes used in histochemistry became bound to cell-specific receptors. He asked” Why can’t such dyes be toxic for specific organisms?”
1910 looking for something to target Treponema pallidum. Looked at arsenic compounds and on the 606th one tested. Salvarsan- combo of salvation and arsenic. First documented case of a chemicla that could selectively kill pathogens w/o permanently harming the human host.

6. History Gerhard Domagk Drugs are changed in the body ORIGIN: Prontosil
(cont’d)
Gerhard Domagk
Drugs are changed in the body
ORIGIN: Prontosil
(Only active in vivo)
DRUG: Sulfanilamide (1935)
NOBEL: 1939
In 1934, found that protosil cured a fatal streptococcal infection in mice - did not work in test tube.
prontosil  sulfanilamide
didn’t work in tt, but working in animal. Turns out that prontosil was split by a enzymes in animals blood into the first of the “Sulfa drugs”
The cure rates for some diseases were rising and these findings gave impetus to the efforts to purify penicillin.

7. History Alexander Fleming Microbes make antibiotics
(cont’d)
Alexander Fleming
Microbes make antibiotics
ORIGIN: moldy culture plate
DRUG: Penicillin (1928)
NOBEL: 1945
Alexander Fleming
penicillin developed in US in spores from mold on coats of scientists

8. History Selman Waksman Soil Streptomyces make antibiotics
(cont’d)
Selman Waksman
Soil Streptomyces make antibiotics
comes up with definition of antibiotic
ORIGIN: Penicillin development
DRUG: Streptomycin (1943)
NOBEL: 1952
Selman Waksman
streptomycin - bacteria produced antibiotics too. Many began screening soils looking for antibiotics.

9. * There is no perfect drug.
The Ideal Drug*
Selective toxicity: against target pathogen but not against host
LD50 (high) vs. MIC and/or MBC (low)
Bactericidal vs. bacteriostatic
Favorable pharmacokinetics: reach target site in body with effective concentration
Spectrum of activity: broad vs. narrow
Lack of “side effects”
Therapeutic index: effective to toxic dose ratio
Little resistance development
Selective toxicity - greater harm to microbes than host, done by interfering with essential biological processes common in bacteria but not human cells.
LD50 vs. MIC - Therapeutic index (the lowest dose toxic to the patient divided by the dose typically used for therapy). High TI are less toxic to the patient.
Bactericidal vs. bacteriostatic
Static rely on normal host defences to kill or eliminate the patogen after its growth has been inhibited. (UTIs) CIDAL given when host defenses cannot be relied on to remove or destroy pathogen.
Favorable pharmacokinetics - drug interxns, how drug is distributed, metabolized and excreted in body (unstabel in acid, can it cross the Blood-brain barrier, etc)
Spectrum of activity
broad spectrum - wide
Narrow spectrum - narrow range (pathogen must be ID’d)
Lack of “side effects” allergic, toxic side effects, suppress normal flora
Little resistance development
* There is no perfect drug.

12. Susceptibility Tests 1. Broth dilution - MIC test
Agar dilution, not used much, pour plates, tilt sideways
2. Agar dilution - MIC test

13. Minimal Inhibitory Concentration (MIC) vs.
Minimal Bactericidal Concentration (MBC)
32 ug/ml
16 ug/ml
8 ug/ml
4 ug/ml
2 ug/ml
1 ug/ml
Sub-culture to agar medium
MIC = 8 ug/ml
MBC = 16 ug/ml

14. Susceptibility Tests Agar diffusion  Kirby-Bauer Disk Diffusion Test
(cont’d)
Agar diffusion
 Kirby-Bauer Disk Diffusion Test

15. Susceptibility Tests “Kirby-Bauer Disk-plate test”
(cont’d)
Diffusion depends upon:
Concentration
Molecular weight
Water solubility
pH and ionization
Binding to agar
Standardized tests
MH media, exact conc. of specific organisms

16. Susceptibility Tests “Kirby-Bauer Disk-plate test”
(cont’d)
Zones of Inhibition (~ antimicrobial activity) depend upon:
pH of environment
Media components
Agar depth, nutrients
Stability of drug
Size of inoculum
Length of incubation
Metabolic activity of organisms
pH of environment - some druga are more activity at high; some at low pH
Media components - Agar depth, nutrients
- some anionic detergents inhibit aminoglycosides; serum protiens can bind penicillin, etc. We always yse same media (Mueller Hinton)
Stability of drug -chlortracycline can be iinactivated at 37; while some aminogylcosides are stable for very long time
Size of inoculum - in general, the larger the inoculum, the lower the “apparent susceptibility” of the organism. Large bacterial populations are less promptly and completely inhibited than small ones. In addition, a resistant mutant is much more likely to emerge in large populations.
Length of incubation - The longer the incubation continues, the greater the chance for resistant mutants to emerge or for the least susceptible members of population to begin growing as drug deteriorates.
Metabolic activity of organisms - Actively growing bacteria are more suseptible than those in resting phase.

17. Antibiotic Mechanisms of Action
Alteration of Cell Membrane Polymyxins Bacitracin Neomycin
Transcription
Translation
Translation

18. Mechanism of Action ANTIMETABOLITE ACTION Sulfonamides
an analog of PABA, works by competitive inhibition
Trimethoprim-sulfamethoxazole
a synergistic combination; useful against UTIs
broad spectrum, cidal?
Very few that do this.
Most useful are folate inhibitors sulfonamide and trimethoprim. Inhibit various steps in the pathway for folic acid that ultimately blocks the synthesis of a coenzyme required for nucleotide synthesis. Animal cells lack the enzymes in the folic acid synthesis portion of the pathway which is why folic acid is a dietary requirement.
Difference in the activity of the various sulfonamides reflect their ability to compete with PABA for the enzyme. High urine levels are achieved and is excreted in urine in active form therefore good for UTIs.

19. Mechanism of Action ANTIMETABOLITE ACTION
(cont’d)
Sulfonamides, which resemble p-aminobenzoic acid and dapsone (leprosy and pneumocytis) competitively inhibit dihydropterate synthase.
Trimethoprim inhibits the enzymatic action of dihydrofolate reductase.
Both of these actions cause interference with the synthesis of folic acid, which is required by bacteria.
tetrahydrofolic acid

20. Mechanism of Action 2. ALTERATION OF CELL MEMBRANES
(cont’d)
2. ALTERATION OF CELL MEMBRANES
Polymyxins and colistin
destroys membranes
active against gram negative bacilli
serious side effects
used mostly for skin & eye infections
Tend to be very toxic.
Have a cationic detergent effect, bind to cell membranes of Gm neg and alter permeability. Toxicity due to the effect that it has on cell membranes as well.
USed topically and have the advantage that resistance to them rarely develops
nephrotoxic and neurotoxic

21. Mechanism of Action ALTERATION OF CELL MEMBRANES
(cont’d)

22. Mechanism of Action INHIBITION OF PROTEIN SYNTHESIS:
(cont’d)
INHIBITION OF PROTEIN SYNTHESIS:
Steps in synthesis:
Initiation
Elongation
Translocation
Termination
80S vs. 70 S But similar to mitochondrial ribosomes which may account for some toxicity
Different aspects of protein synthesis are impacted on based on the target.
Prokaryotes and eukaryotes (80S) have a different structure to ribosomes so can use antibiotics for selective toxicity against ribosomes of prokaryotes (70S)

23. Mechanism of Action INHIBITION OF PROTEIN SYNTHESIS
(cont’d)
Aminoglycosides
bind to bacterial ribosome on 30S subunit; and blocks formation of initiation complex. Both actions lead to mis-incorporation of amino acids
Examples:
Gentamicin Tobramycin
Amikacin Streptomycin
Kanamycin Spectinomycin
Neomycin
Tend to be bactericidal and broad spectrum
Are actively transported onto the bacterial cell (this is one reason for selective toxicity - don’t work against animal cells) by a mechanism that involves ox phos. Therefore, they have little or no activity against strict anaerobes or those that metabolize only fermentatively (like streptococci)
Gentamicin
Tobramycin
Amikacin
Streptomycin - hardly used anymore b/c high-level and stable resistant mutants are frequently selected for during therapy
Kanamycin
Spectinomycin
Neomycin - used to reduce the facultative flora of the large intestine befroe certain types of intestinal surgury. It is poorly absorbed therefore is active in the bowel.

24. Mechanism of Action INHIBITION OF PROTEIN SYNTHESIS
(cont’d)
Aminoglycosides (cont’d)
broad spectrum
Gram negative rods
P. aeruginosa
Drug-resistant gram negative rods
Plague, Tularemia, Gonorrhea
Pre-op (bowel)
External (skin)
toxic at some level to eighth cranial nerve
Eighth cranial nerve (kanamycin and amikacin - hearing)
Neomycin - used to reduce the facultative flora of the large intestine befroe certain types of intestinal surgury. It is poorly absorbed therefore is active in the bowel.
Lac of activity angainst mostly anaerobic envirnoment of large bowel may account low level side effect. for

25. Mechanism of Action INHIBITION OF PROTEIN SYNTHESIS
(cont’d)
Macrolides: chloramphenicol & erythromycin
bind to 50S subunit and blocks the translocation step
Anaerobes
Typhoid
Meningitis
Chloramphenicol: broad spectrum
Erythromycin:
Most macrolides tend to be bacteristatic (cidal for some Gm +)
Chloramphenicol: good permeability across blood-brain barrier but does cause some toxicity, metabolized in liver and low levels in urine.
Other macrolides are: azithromycin
clarithromycin
Mycoplasma
Legionella
S. pyogenes

26. Mechanism of Action INHIBITION OF PROTEIN SYNTHESIS
(cont’d)
Clindamycin
binds to 50S subunit and interferes with binding of the amino acid – acyl-tRNA complex and so inhibits peptidyl transferase
works best against
Staphylococcus
Bacteroides & anaerobic gram neg rods
Penicillin allergic people
similar mode of action as macrolides

27. Mechanism of Action INHIBITION OF PROTEIN SYNTHESIS
(cont’d)
Tetracyclines
bind to 30S subunit and interferes with the attachment of the tRNA carrying amino acids to the ribosome
effective against:
Chlamydia
Rickettsia
Mycoplasma
Brucella
Mostly bacteriostatic
broad spectrum
Active against obligate intracellular bacteria and cell wall deficient organisms
chelated by divalent cations and their absorption and activity are reduced, so shouldn’t be taken with dairy products or many antacids
used in otis medea and sinusitis b/c usually effective, but also given in viral cases, which is bad. Probably no antibiotic group has been oversubscribed more.
Doxycyline - longer acting tetracyline

28. Mechanism of Action 4. INHIBITION OF DNA/RNA SYNTHESIS Rifampin
(cont’d)
4. INHIBITION OF DNA/RNA SYNTHESIS
Rifampin
binds to RNA polymerase
active against gram positive cocci
bactericidal for Mycobacterium
used for treatment and prevention of meningococcus
use along with other drugs for TB

29. Mechanism of Action INHIBITION OF DNA/RNA SYNTHESIS
(cont’d)
Metronidazole
breaks down into intermediate that causes breakage of DNA
active against:
protozoan infections
anaerobic gram negative infections
Metronidazole used in treating trichomonas, giardia and amebic infections and some anaerobes, like bacteroides
First one was nalidixic acid, but it had limited use because therapeutic levels were only attained in urine and there was high level mutational resistance developed VERY Rapidly.
Fluorine enhances activity against Gm neg and adds activity against Gm pos.
Quinolones and fluoroquinolones
effect DNA gyrase
broad spectrum

30. Mechanism of Action INHIBITION OF DNA/RNA SYNTHESIS
(cont’d)

31. Mechanism of Action CELL WALL SYNTHESIS INHIBITORS Steps in synthesis:
(cont’d)
CELL WALL SYNTHESIS INHIBITORS
Steps in synthesis:
NAM-peptide made in cytoplasm
attached to bactoprenol in cell membrane
NAG is added
whole piece is added to growing cell wall
crosslinks added
the β-Lactams
the non β-Lactams

33. Mechanism of Action CELL WALL SYNTHESIS INHIBITORS
(cont’d)
CELL WALL SYNTHESIS INHIBITORS
β-Lactam Antibiotics
Penicillins
Cephalosporins
Carbapenems
Monobactams
Penicillins
penicillin,
oxacillin
ampicillin
Cephalosporins & Cephamycins
cefaclor
cefoxitin
cephalosporin
Carbapenems
Imipenem
Monobactams
moncyclic (different from other b-lactams)
Axtreonam

34. Mechanism of Action CELL WALL SYNTHESIS INHIBITORS
(cont’d)
β-Lactam ring structure
High therapeutic index
some block NAM elongation - Vancomycin
precursor transport blockage - Bacitracin
β-lactam drugs
beta lactam ring has structural similarity between normal substrate (for enzymes known as penicillin binding proteins (PBPs) By mimicking the substrate the beta lactam drugs are bound by PBPs thus competitively inhibiting their enzymatic activity. This causes a disruption in CW synthesis. B/c CW are only synthesized in actively multiplying cells, these are only effective against growing bacteria.
Some make beta lactamse, an enzyme that breaks down the ring structure and thus inactivates penicillins

36. Mechanism of Action CELL WALL SYNTHESIS INHIBITORS
(cont’d)
Action of β-Lactam antibiotics
Bactericidal; growing cells only
Drug links covalently to regulatory enzymes called PBPs (penicillin-binding proteins)
Blocks cross-linkage of peptidoglycan

37. Mechanism of Action CELL WALL SYNTHESIS INHIBITORS
(cont’d)
Action of β-Lactam antibiotics
For E. coli
> MIC
wall damage
autolysins
spheroplasting
cell lysis
< MIC
no septa
filaments

39. Mechanism of Action CELL WALL SYNTHESIS INHIBITORS
(cont’d)
Resistance to β-Lactams – Gram pos.

40. Mechanism of Action CELL WALL SYNTHESIS INHIBITORS
(cont’d)
Resistance to β-Lactams – Gram neg.

41. Mechanism of Action CELL WALL SYNTHESIS INHIBITORS
(cont’d)
Non - β-Lactams
Vancomycin
active against gram positive cocci, but not gram negative because too large to pass through outer membrane
interferes with PG elongation
Cycloserine, ethionamide and isoniazid
inhibits enzymes that catalyze cell wall synthesis
for Mycobacterial infections

42. Clinical Uses PATHOGENS TYPICAL DRUG Gram positive Pen-ase (-)
Penicillin G (oral or IM)
Methicillin, Nafcillin
Gram negative
Enterics, etc.
Pseudomonas
B. fragilis
Ampicillin, gentamicin, etc.
Ticarcillin, tobramycin
Clindamycin

43. Iso-nicotinic hydrazide (INH) Amphotericin B, ketoconazol
Clinical Uses
(cont’d)
PATHOGENS
TYPICAL DRUG
Mycobacterium
Streptomycin
Iso-nicotinic hydrazide (INH)
Fungi:
Cutaneous
Deep
Nystatin
Amphotericin B, ketoconazol
Parasites:
Plasmodium
Giardia
Chloroquine
Quinacrine

44. Resistance Physiological Mechanisms
1. Lack of entry – tet, fosfomycin
2. Greater exit
efflux pumps
tet (R factors)
3. Enzymatic inactivation
bla (penase) – hydrolysis
CAT – chloramphenicol acetyl transferase
Aminogylcosides & transferases

45. Resistance Physiological Mechanisms
(cont’d)
4. Altered target
RIF – altered RNA polymerase (mutants)
NAL – altered DNA gyrase
STR – altered ribosomal proteins
ERY – methylation of 23S rRNA
5. Synthesis of resistant pathway
TMPr plasmid has gene for DHF reductase; insensitive to TMP

46. Origin of Drug Resistance
Non-genetic
metabolic inactivity
Mycobacteria
non-genetic loss of target
penicillin – non-growing cells, L-forms
intrinsic resistance
some species naturally insensitive

47. Origin of Drug Resistance
(cont’d)
Genetic
spontaneous mutation of old genes
Vertical evolution
Acquisition of new genes
Horizontal evolution
Chromosomal Resistance
Extrachromosomal Resistance
Plasmids, Transposons, Integrons
Spontaneous evolution occurs at low rate (~1 in 10 million cells)
Grow up 10 to the 9 or tenth cells, there is a good chance one cell is resistant to Streptomycin due to mutation, plate the load, isolate the resistant.
Streptomycin: binds to 30S subunit of ribsome
causes distortion and misreading
Single target, easy to get spontaneuos mutation. Multiple targets are harder b/c several different mutations are required to prevent binding of the drug.
Plasmids
Transposons
Integrons
Thus if organism has two different plasmids, an antibiotic resistant gene can move from one to another. In this way, a gene could move from a narrow range host plasmid to a wide range host plasmid. Wide range host plasmids, in contrast to narrow range host plasmids, can replicate even if they are transferred to unrelated bacteria.

48. Plasmids independent replicons dispensable several genes host range
circular DNA
dispensable
several genes
drug resistance
metabolic enzymes
virulence factors
host range
restricted or broad

49. Plasmids size Transfer between cells: small, non-conjugal
(cont’d)
size
small, non-conjugal
large, conjugal <25 kbp
Transfer between cells:
CONJUGATION (cell to cell contact)
due to plasmid tra genes (for pili, etc)
NON-CONJUGAL
transduction
mobilization by conjugation plasmids
transduction – Staph bla

50. Implications of Resistance
Household agents
they inhibit bacterial growth
purpose is to prevent transmission of disease-causing microbes to noninfected persons.
can select for resistant strains
NO evidence that they are useful in a healthy household

51. Implications of Resistance
Triclosan studies
effect diluted by water
one gene mutation for resistance
contact time exceeds normal handwash time (5 seconds)
Allergies
link between too much hygiene and increased allergy frequency

52. Implications of Resistance

54. REVIEW

55. Minimal Inhibitory Concentration (MIC) vs.
Minimal Bactericidal Concentration (MBC)
32 ug/ml
16 ug/ml
8 ug/ml
4 ug/ml
2 ug/ml
1 ug/ml
Sub-culture to agar medium
MIC = 8 ug/ml
MBC = 16 ug/ml
REVIEW

56. What are main targets of Antibiotics?
REVIEW

57. Mechanism of Action INHIBITION OF CELL WALL SYNTHESIS β-Lactams
Non β-Lactams
REVIEW

58. Mechanism of Action CELL WALL SYNTHESIS INHIBITORS
(cont’d)
β-Lactam ring structure
High therapeutic index
some block NAM elongation - Vancomycin
precursor transport blockage - Bacitracin
β-lactam drugs
beta lactam ring has structural similarity between normal substrate (for enzymes known as penicillin binding proteins (PBPs) By mimicking the substrate the beta lactam drugs are bound by PBPs thus competitively inhibiting their enzymatic activity. This causes a disruption in CW synthesis. B/c CW are only synthesized in actively multiplying cells, these are only effective against growing bacteria.
Some make beta lactamse, an enzyme that breaks down the ring structure and thus inactivates penicillins
REVIEW

59. Mechanism of Action INHIBITION OF PROTEIN SYNTHESIS Aminoglycosides
Macrolides
Chloramphenicol
Erythromycin
Tetracyclines
Clindamycin
Tend to be bactericidal and broad spectrum
Are actively transported onto the bacterial cell (this is one reason for selective toxicity - don’t work against animal cells) by a mechanism that involves ox phos. Therefore, they have little or no activity against strict anaerobes or those that metabolize only fermentatively (like streptococci)
Gentamicin
Tobramycin
Amikacin
Streptomycin - hardly used anymore b/c high-level and stable resistant mutants are frequently selected for during therapy
Kanamycin
Spectinomycin
Neomycin - used to reduce the facultative flora of the large intestine befroe certain types of intestinal surgury. It is poorly absorbed therefore is active in the bowel.
REVIEW

60. INHIBITION OF NUCLEIC ACID SYNTHESIS
Mechanism of Action
INHIBITION OF NUCLEIC ACID SYNTHESIS
Rifampin
Metronidazole
Quinolones and fluoroquinolones
use along with other drugs for TB
REVIEW

61. Mechanism of Action DISRUPTION OF CELL MEMBRANES Polymyxins Colistin
Tend to be very toxic.
Have a cationic detergent effect, bind to cell membranes of Gm neg and alter permeability. Toxicity due to the effect that it has on cell membranes as well.
USed topically and have the advantage that resistance to them rarely develops
nephrotoxic and neurotoxic
REVIEW

62. Mechanism of Action ANTIMETABOLITE ACTION Sulfonamides
Trimethoprim-sulfamethoxazole
broad spectrum, cidal?
Very few that do this.
Most useful are folate inhibitors sulfonamide and trimethoprim. Inhibit various steps in the pathway for folic acid that ultimately blocks the synthesis of a coenzyme required for nucleotide synthesis. Animal cells lack the enzymes in the folic acid synthesis portion of the pathway which is why folic acid is a dietary requirement.
Difference in the activity of the various sulfonamides reflect their ability to compete with PABA for the enzyme. High urine levels are achieved and is excreted in urine in active form therefore good for UTIs.
REVIEW

63. Resistance Physiological Mechanisms
1. Lack of entry – tet, fosfomycin
2. Greater exit
efflux pumps
tet (R factors)
3. Enzymatic inactivation
bla (penase) – hydrolysis
CAT – chloramphenicol acetyl transferase
Aminogylcosides & transferases
REVIEW

64. Resistance Physiological Mechanisms
(cont’d)
4. Altered target
RIF – altered RNA polymerase (mutants)
NAL – altered DNA gyrase
STR – altered ribosomal proteins
ERY – methylation of 23S rRNA
5. Synthesis of resistant pathway
TMPr plasmid has gene for DHF reductase; insensitive to TMP
REVIEW

65. Mechanism of Action CELL WALL SYNTHESIS INHIBITORS
(cont’d)
Resistance to β-Lactams – Gram pos.
REVIEW

66. Mechanism of Action CELL WALL SYNTHESIS INHIBITORS
(cont’d)
Resistance to β-Lactams – Gram neg.
REVIEW