ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
I. History
A. Natural products and folk medicine
Cinchona bark -quinine
B. Rational approach to chemotherapy
Ehrlich - magic bullet
Domagk - therapeutic effectiveness of sulfonamide
Fleming - penicillin
Florey et al. - produced penicillin in sufficient quantities; demonstrated clinical effectiveness
II. Properties of antimicrobial agents
A. Selective toxicity (Figure 2, Figure 3, Figure 4, Figure 5)
Bacterial cell wall; bacterial ribosome
Definitions:
antimicrobial agents
antibiotics
bactericidal, bacteristatic (viricidal, fungicidal)
Possible targets for drug therapy
1. pathways not found in host
a. folic acid synthesis
sulfanilamide competitively inhibits the incorporation of PABA into folic acid (antimicrobial)
b. cell wall synthesis - host cells don't have comparable structure
penicillins
cycloserine
vancomycin
bacitracin
2. Host pathway is less sensitive
50% inhibitory concentration for trimethoprim (blocks reduction of dihydrofolate to tetrahydofolate) is 5 µM for bacteria but 250 mM for mammals
3. Microorganism concentrates the drug tetracycline
4. Pathway is present but is significantly different
review flow of information in the cell and points of antibiotic attack
Interactions among drugs (Figure 7)
synergism - sulfa and trimethoprim
antagonism - penicillin and chloramphenicol
neutral
B. Ideal antimicrobial agent: Narrowest sprectrum with fewest side effects and lowest toxicity
C. Aim of chemotherapy
1. Get enough drug to infected tissue to inhibit or kill pathogen
a. Influenced by pharmacological properties of antibiotic
Some antibiotics absorbed well enough after oral administration to treat systemic infections
Some concentrated in urine - can give small oral doses to achieve high concentration in urine
If poorly absorbed or degraded in GI tract must administer parenterally.
After absorption most abs circulate bound to plasma proteins (especially albumins)
Concentration in tissues and serum depends on degree of protein binding and ability to diffuse in to tissues.
D. Factors considered when treating with antibiotics (in addition to above)
1. Accurate clinical diagnosis of site and extent of infection
Skin infection ( topical application)
Walled off abscess (Best treatment may be drainage - hard to get antibiotic to site of this type of infection)
2. Overall immune status of patient
Poorer results achieved in compromised hosts. **this reflects the decrease contribution of the host's immune response
There is greater risk of superinfection in these patients
3. Underlying physical condition of patient
Age
Pregnant or nursing-many abs can cross placenta, appear in milk
Metabolic disorders-diabetes
Gastric Acidity-affects oral absorption
Renal or hepatic disease-impaired renal function can lead to decreased excretion and thus higher concentration of drugs in circulation
4. Identity of organism and results of susceptibility testing
Therapy begun before identity known - adjusted when results from lab available
(This emphasizes importance of direct smear results in selection of initial therapy)
5. Cost
E. MEASURE OF INVITRO ACTIVITY OF ANTIMICROBIAL AGENT
1. Bacteriocidal and bacteriostatic activity (Figure 9)
2. Measure of activity
MIC
MBC
3. Lab determination of MIC and MBC (Figure 10)
Tube test, Kirby Bauer
Highly standardized
Tissue levels required to inhibit/kill (ie. must exceed MIC)
4. Importance of MIC/MBC to physician
MIC
Must achieve MIC at site of infection to inhibit
Concentrations higher than MIC used to account for serum binding and other factors
MBC
Impt for patience with lowered immune responses
Must know concentration which will kill infecting organisms
OVERALL IMMUNE STATUS OF PATIENT
Poorer results achieved in compromised hosts. `This reflects not a lesser activity of drug but rather a decreased contribution by hosts immune response
5. Which organisms are tested for antimicrobial susceptibility
a. Tests done on pathogens whose susceptibility cannot be predicted from knowledge of their identity (e.g. S. aureus, S. epidermidis;Enterobacteriaceae; Pseudomonas sp.; N. gonorrhoeae; H. influenzae )
b. Some gps not tested; eg. S. pyogenes, N. meningiditis
6. Single vs combined therapy
a. Likely development of resistance
b. Overwhelming infection - need more effective killing
c. Polymicrobic infection - different classes of mo's involved and can't treat with a single agent
d. Poor health status of patient
e. Must consider drug interactions
Synergy, antagonism, Indifference
III. Mechanism of action of antimicrobial drugs
A. Inhibition of cell wall synthesis
B. Alteration of cell membrane permeability
C. Inhibition of protein synthesis
D. Inhibition of nucleic acid synthesis
A. Inhibition of cell wall synthesis
1. Bacterial cell wall
Rigid
High internal osmotic pressure: (Gm+ 3-5x higher than Gm-)
Injury to wall (eg.lysozyme) or inhibition of its formation may lead to lysis
2. Antibiotics affecting cell wall: e.g. penicillin, cephalosporins, bacitracin, vancomycin
3. Peptidoglycan (found only in bacteria)
Polysaccharide (NAM & NAG)
Polypeptide
Steps in synthesis of PG and insertion into growing wall
4. Penicillins and cephalosporins
Inhibit cell wall synthesis
Role of PBPs (transpeptidation)
Lysis of cell (role of autolytic enzymes0
5. Increased spectrum penicillins
Problems with Gm-s and penicillin
Benzyl penicillin (G)
Semisynthetic penicillin: Methacillin, Nafcillin -side chain protects -lactam ring
Extended-spectrum penicillins: ampicillin, amoxicillin
Increased activity against Gm- organsims-can penetrate outer membrane
6. Other drugs
Bacitracin, Vancomycin inhibit early steps in PG synthesis (within membrane - so these drugs must penetrate membrane
B. Inhibition of cell membrane function
1. Cytoplasm bounded by CM
a. Selective permeability
b. Carries out active transport
disruption of functional integrity permits escape of ions and macromolecules -->damage or death
c. CM of bacteria and fungi different from animal cells.
More readily disrupted by certain agents
POLYMYXINS: act selectively on membranes rich in phosphotidylethanolamine
Acts like cationic detergent
Doesn't act on membranes containing sterols
(eg. fungi and animal cells)
POLYENE ANTIBIOTICS: Amphoterecin active against fungi aqueous pores in CM
Act on membranes rich in sterols (esp. ergosterol which is found in CM of fungi)
Inactive against bacteria because no sterols in membranes
C. Inhibition of protein synthesis
1. Review of protein synthesis and differences between bacteria and human machinery
2. Antiribosomal antibiotics
a. Aminoglycosides - bacteriocidal (most others bacteriostatic)
b. Some act selectively on prokaryotic ribosomes, some on cytoplasmic ribosomes of eukaryotes, mitochondrial ribosomes respond like procaryotic ribosomes
Puromycin - Eu & Pro -(Lg) Releases peptidyl-puromycin
Tetracycline - Pro -(S) Blocks stable binding in A site
Streptomycin - Pro -(S) Blocks movement of initiation complex
Causes misreading
c. Some act on polysomal ribosomes (CAM, puromycin), some block only free, initiating ribosomes (binding sites accessible)
CAM - Pro - (L)Blocks peptidyl transfer
d. Some bind to small and other to large ribosomal subunit
D. Inhibition of nucleic acid synthesis
1. Examples:
Nalidixic Acid - Blocks DNA gyrase
Rifampin - Transcription; Inhibits RNA synthesis -Binding to DNA- dependent RNA polymerase of bacteria
Sulfonamides - -> Inhibits Folic Acid synthesis --> Nucleic acid synthesis
Bacteria synthesize own FA
Humans require preformed FA
Trimethoprim - Inhibits folic acid metabolism --> Nucleic acid synthesis
Why are there a broader array of drugs to treat bacterial infections than fungal or viral infections?
IV. Antifungal agents
Polyenes - Amphotericin B
binds more tightly to ergosterol (found in fungal membranes) than to cholesterol (found in higher eucaryotes) -> membrane damage
Griseofulvin -
concentrates in skin and nails so can use for superficial fungal infections
V. Antiviral agents
amantadine - interferes with uncoating of influenza A, some effects on other viruses
acyclovir - DNA polymerase inhibitor, must be phosphorylated by thymidine kinase, has a higher affinity for the viral (herpes) enzyme than the mammalian enzyme
ribavirin - purine analog
azidothymidine - nucleotide analog
A. Why are there so few antiviral agents? (Figure 11)
B. Steps in replication representing potencial drug targets (Figure 12)
C. Approaches
RESISTANCE TO ANTIMICROBIAL DRUGS
A. General
a. Produce enzymes that inactivated drug
b-lactamases-cleave b-lactam ring of penicillin and cephalosporins adenylating, phosphorylating,acetylating enzymes
b. Change permeability to drug
Tetracyclines accumulate in susceptibile bacteria not in resistant bacteria.
c. Develop altered structural target for drug.
Aminiglycoside resistance-->altered protein in 30S subunit of ribosome
d. Develop altered metabolic pathway to bypass inhibited reaction.
Some sulfonamide resistant bacteria can utilize preformed folic acid
e. Develop altered enzyme that can still perform metabolic function but less affected than enzyme in susceptible organism
Change in affinity for enzyme for sulfonamide that for PABA
2. Most drug resistance due to genetic change in organism
Either chromosomal mutation of acquition of plasmid or transposon.
B. Genetic Basis of Resistance (Figure 15)
1. Chromosome-mediated resistance
a. Mutation in gene coding for either target of drug or transport system in membrane that controls uptake of drug
b. Frequency of spontaneous mutation ~10-7 to 10-9 ; this is much lower than frequency of acquition of resistance plasmids.
Chromosomal resistance is less of a clinical problem than is plasmid-mediated resistance
2. Plasmid-Mediated Resistance
Important from clinical point of view for 3 reasons
a. Occurs in many different species (especially Gm- rods)
b. Plasmids frequently mediate resistance to multiple drugs
c. Plasmids have a high rate of transfer from one cell to another (usually by conjugation) (Figure 16)
Resistance Plasmids (resistance factors, R factors)
Impt. properties:
Can replicate independently of bacterial chromosome, so a cell can contain many copies
Can be transferred to cells of other species and genera
Other traits imparted by R factors
Resistance to metal ions, eg. mercuric ions --> reduced to elemental mercury (Impt in clinical setting as Hg++ is active ingredient of merthiolate.
3. Transposon-mediated Resistance
a. Transposons are genes that are transferred either within or between larger pieces of DNA such as the bacterial chromosome and plasmids
b. Typical drug resistant transposon composed of 3 genes flanked on both sides by shorter DNA sequences --usually a series of inverted repeats that mediate the interaction of the transposon with the larger DNA
c. 3 genes code for :
Transposase -catalyzes excision and reintergrating of the transposon
Represson - regulates synthesis of transposase
Drug resistance gene
4. Transmission of resistance determinants
a. Spontaneous mutations
b. Transformation (Chrm or plasmid)
c. Conjugation
d. Transduction
C. Specific Mechanisms of Resistance (Figure 17, Figure 17A)
Penicillins:
b-lactamases (most on plasmids)
Lack of penicillin receptors (PBPs) or inaccessibility of receptors because of permeability barriers of outer membrane (These are ofter under chromosomal control)
Failure of activation of autolytic enzymes in cell wall - can inhibit growth without killing
Failure to synthesize peptidoglycan (mycoplasmas, L-forms, non-growing bacteria
Aminoglycosides:
Modification of drug by plasmid-encoded phosphorylating, adenylating, and acetylating enzymes (most inportant mechanism)
Chromosomal mutation in gene that codes for target protein in 30S subunit of bacterial ribosome
Decreased permeability of the cell to the drug and lack of active transport into the cell (can be chromosomal or plasmid)
Tetracyclines:
Resistance result of failure of drug to reach an inhibitory concentration inside the bacteria (plasmid-encoded process that either reduces uptake of drug or enhances its transport out of cell)
Chloramphenicol:
Plasmid encoded acetyltransferase - acetylates drug --> inactivates it.
Erythromycin:
Plasmid-encoded enzyme methalytes the 23S ribosomal RNA thereby blocking binding of drug
Sulfonamides:
Plasmid-encoded transport system actively exports drug out of cell
Chromosomal mutation in gene coding for target enzyme - reduces binding affinity of drug
Rifampin:
Chromosomal mutation in gene for the subunit of the bacterial RNA polymerase results in ineffective binding of the drug
D. Nongenetic Basis for Resistance (Figure 18)
1. Bacteria can be walled off within an abscess which drug cannot penetrate
2. Bacteria can be in resiting state --ie. not growing - therefore insensitive to cell wall inhibitors such as penicillin.
3. Under certain circumstances, organisms that would ordinarly be killed by penicillin can lose their cell walls and survive as protoplasts and bew insesitive to cell-wall-active drugs.
4. Several artifacts can make it appear that the organisms are resistant
Administration of wrong drug or wrong dose
Failure of drug to reach appropriate body site
Improper administration of drug
Failure of patient to take drug
E. Selection of Resistant Bacteria by Overuse and Misuse of Antibiotics (Figure 19)
3 Main Points
1. Some physicians:
a. Use multiple antibiotics when one would be sufficient
b. Prescribe unnecessarily long courses of antibiotic therapy
c. Use antibiotics in self-limited infections for which they are not needed
d. Overuse antibiotics for prophylaxis before and after surgery
2. In many countries antibiotics are sold over the counter to the general public - this encourges inappropriate and indiscriminant use of the drugs
3. Antibiotics are used in animal feed to prevent infections and promote growth:
This selects for resistant organisms in the animals and may contribute to the pool of resistant organisms in humans
F. Control of Resistance (Figure 20, Figure 20A)
