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Antibiotics
• Hugh B. Fackrell
• Filename: antibiot.ppt
4/13/2015
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Outline
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History
Ideal properties
Sources
“Sulfas”
– Antimetabolites
– antibiotic synergism
• Major Groups of antibiotics
• Mechanisms of action
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History
• Salvarsan 606
• Prontosil
• Penicillin
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Salvarsan 606
• Paul Ehrlich
– early 1900’s
– syphilis
– arsenic + organic compound
• Aniline dyes -
– wasn't able to find the "magic bullet”
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Prontosil
• 1930's, Gerhard Domagk
– Prontosil
• 1935, Jacques and Therese Trefoncel
– discovered that the active compound in
Prontosil was Sulfanilamide
• sulfanilamide “ Sulfas”
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Penicillin
• 1928, Alexander Fleming
– antibacterial activity in Penicillium mold
(called it Penicillin)
• 1938, Howard Florey and Ernst Chain
– developed Penicillin as an effective antibiotic
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Antimicrobial Therapy
• Antimicrobics
– substances produced by microbes that inhibit
other microbes
• Semi-synthetic antibiotics
– naturally produced but altered
• Synthetic antibiotics:
– derived from chemicals
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Ideal Properties of an Antibiotic
• Low toxicity for patient
– kills the invading microorganism without
damaging the host
– no adverse side reactions
– non allergenic
• High toxicity for microbe
– bactericidal not bacteriostatic
– broad spectrum
• Low risk of other infections
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More Characteristics
• drug can be administered orally or
parenterally (by injection)
– Soluble in tissue fluids
– absorbed by and dissolved in tissues or body
fluids
• levels of active drug sustained long enough
to kill the invading agent
• Long “Shelf” life
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Still More Characteristics
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Low probability of resistance
Microbial drug resistance develops slowly
microbicidal rather than microbistatic
Not inactivated by organic material
Assists the host in eliminating the infecting
microbe
• Not a powerful allergen
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Sources of Antibiotics
Most spore-forming microorganisms
• Fungi
– Penicillium penicillin,
– Cephalosporium griseofulvin
• Bacteria
– Bacillus bacitracin, polymyxin, tyrothricin,
colimycin, gramicidin
– Streptomycetes Aminoglycosides, nystatin,
chloramphenicol, erythromycin, tetracylcine...
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Mechanisms of Drug Action
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inhibit cell wall synthesis
inhibit nucleic acid synthesis
inhibit protein synthesis
interfere with cell membrane function
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Sulfa Drugs
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Sulfa vs PABA
NH2SO2
NH2
Sulfanilamide
NH2
HOOC
PABA
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Structure of Sulfa Drugs
Sulfisoxazole
Sulfanilamide
Prontosil
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Folic Acid Metabolism
PABA + pteridine
Sulfonamide
[GTP]
Pteridine
synthetase
Dihydropteroic acid
Dihydrofolate
L- Glutamine
Synthetase
Dihydrofolic Acid
2 NADPH
Trimethoprim
2 NADP+
Dihydrofolate
synthetase
Tetrahydrofolic Acid
Thymidine
DNA
Purines
DNA, RNA
Methionine
tRNa, 4/13/2015
Proteins
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Folic Acid Inhibition
PABA + pteridine
Sulfonamide
Dihydropteroic acid
Dihydrofolic Acid
Trimethoprim
Tetrahydrofolic Acid
Thymidine
DNA
Purines
DNA, RNA
Methionine
tRNa, Proteins
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Antibiotic Synergism
Sulfisoxazole
Trimethoprim
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Antibiotic Synergism
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Sulfonamide + trimethoprim
Effective dosage 10% of two separately
Broader spectrum of action
Reduce emergence of resistant strains
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Major Groups of Antibiotics
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Major Groups of Antibiotics
• Aminoglycosides
– streptomycin, kanamycin, neomycin,
gentamicin, spectinomycin, tobramycin,
amikacin
• Beta lactams
– Penicillins, cephalosporins
• Lincomycins
– lincomycin clindamycin
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Major Groups of Antibiotics
• Macrolides
– erythromycin, carbomycin
• Polypeptides
– polymyxin, colimycin, bacitracin, tyrothricin
• Polyenes
– amphotericin B, nystatin
• Rifamycins
– Rifampin
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Major Groups of Antibiotics
• Synthetic
– pyridine
• isoniazid, ethambutol
– sulfonamides
• sulfanilamide, sulphisoxazole
– misc
• nitrofurans, metronidazole, nalidixic acid
• Tetracyclines
– oxytetracline, chlortetracycline
• Unclassified
– Chloramphenicol, vancomycin
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PENEMS
• Carbapenems
• “Ideal” antibiotics
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non toxic
broad spectrum
good “Shelf” life
effective at very low conc
• Attach to Penicillin Binding Proteins
– found in cell membrane
– Gm+ve lysis through loss of cell wall integrity
– Gm -ve filamentous bacteria loss of septum formation
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Adverse Effects of Antibiotics
• Aminoglycosides
– Ototoxic- destroys
cochlear hair cells
– renal toxic
• Chloramphenicol
– depresses bone marrow
– aplastic anemia
– fatal “Grey baby”
syndrome
• Penicillins
– allergy anaphylaxis
• Vancomycin
– thrombophlebitis
– ototoxic
– renal toxic
• Polymyxin, bacitracin
colimycin
– renal toxic
• Sulfas
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skin allergy
anemia
renal toxic
hepato toxic
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Adverse Effects of Antibiotics
• Broad spectrum
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Super infections
Candida albicans
Clostridium difficle
Staphylococcus
Gram -ve
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Tetraclycine
– Depress bone marrow
– “Yellow teeth”
 Pregnant women
 children <7 years
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Mode of Action of Antibiotics
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Mode of Action of Antibiotics
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Inhibit Synthesis of Cell Wall
Damage Cell Membrane
Inhibit Protein Synthesis
Inhibit Nucleic acid Synthesis
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Bacterial Cell Wall
• Peptidoglycan
– many layers in gram positives
– thin in gram negative
• protects the cell against rupture from hypotonic
environments
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Synthesis of peptidoglycan (1/4)
• Uridine diphosphate (UDP) derivatives of
NAM and NAG are synthesized in the
cytoplasm
• Amino acids are sequentially added to
UDP-NAM to form the pentapeptide chain
using ATP as an energy source. The two
terminal D-alanines are added as a dipeptide
(Cycloserine)
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Synthesis of peptidoglycan (2/4)
• The NAM- pentapeptide is transferred from
UDP to a bactoprenol PO4 at the membrane
surface. Bactoprenol is a 55-Carbon alcohol
that attaches to NAM by a pyrophosphate group
and moves peptidoglycan components through
the hydrophobic membrane
• UDP-NAG adds NAG to the NAMpentapeptide to form the peptidoglycan repeat
unit
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Synthesis of peptidoglycan (3/4)
• The completed NAM-NAG peptidoglycan
repeat unit is transported across the
membrane to its outer surface by the
bactoprenol pyrophosphate carrier
• The peptidoglycan unit is attached to the
growing end.
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Synthesis of peptidoglycan (4/4)
• The bactoprenol carrier returns to the inside
of the membrane to collect another NAMpentapeptide. Bactoprenol pyrophosphate
must give up phosphate to connect
Bacitracin
• Finally, transpeptidization - interbridges
formed
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Inhibit Synthesis of Cell Wall
• penicillin, bacitracin, vancomycin,
cephalosporin, carbapenems
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Inhibition of Cell Wall Synthesis
• Cycloserine - inhibits peptidoglycan sub-unit
formation
• Vancomycin - inhibits peptidoglycan elongation
• Beta-lactam antibiotics - Penicillins
– lactam antibiotics block peptidases required to
connect inter bridges
• Cephalosporins bind to the peptidases that are
essential to cross link the glycan molecules.
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Inhibition of cell wall synthesis
• Cycloserine - inhibits the addition of the
two terminal
D-alanines
• Bacitracin - inhibits the transport of the
subunits to their position in the cell wall
• Vancomycin - inhibits the elongation of the
peptidoglycan to form connecting units
Murray 2.4 & 5.4, p. 10
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Inhibition of Cell Wall Synthesis
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Natural Penicillins
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Semi Synthetic Penicillins
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Semi Sythetic Penicillins -2
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Structure of Penicillin
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Hydrolysis of Beta Lactam Ring
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Comparison of Structures
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Pen G in Blood
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Damage Cell Membrane
polymyxin, colimycin, nystatin,
amphoteracin, tyrothricin
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Injury of Plasma Membrane
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Polymyxin action
• Polymyxin B binds to the cell membrane to
disrupts its structural and permeability
properties
Polymyxin
Membrane
Cytoplasm
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Inhibit Protein Synthesis
• Binds to 50S ribosomal subunit
– prevents peptide chain elongation
• clindamycin, chlorampenicol, erythromycin
– block rRNA(23S)
• lincomycin, macrolides
• Binds to 30S ribosomal subunit
– misreading of mRNA
• aminoglycosides- genetamcin
– Blocks binding of tRNA-AA to 30S
• tetraclyclines
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Inhibition of Translation
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Translation
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Inhibition of Peptide Bond
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Inhibition of Ribosome Movement
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Inhibition of tRNA Attachment
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Misreading mRNA
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Inhibit Nucleic acid Synthesis
Quinolones
– Ciprofloxacin and other quinolones
• Inhibits DNA gyrase
• Blocks DNA replication
• Inhibits mitochondrial DNA
– conc in tissues too low for toxicity
• Urinary and intestinal infections
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Inhibition of DNA Replication
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Inhibit Nucleic Acid Synthesis
• Rifamycin
– Inhibits DNA dependent RNA polymerase
– Blocks transcription DNA ->RNA
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Acylovir vs Deoxyguanosine
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Inhibition of Transcription
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Antimetabolites
• Sulfonamides
• Donald D. Woods
• Sulfanilamide blocks folic acid
– folic acid is essential to the synthesis of DNA and
RNA
– Para amino benzoic acid (PABA) not incorporated
into folic acid
• Reversible inhibition
– High [PABA] competitively inhibit sulfanilamide
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Inhibited metabolites,Synthesis
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Drug Resistance
• synthesis of enzymes that inactivate the drug
• decrease in cell permeability and uptake of the
drug
• change in the number or affinity of drug
receptor sites
• modification of an essential metabolic
pathway
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Development of Drug Resistance
• intrinsic
– chromosomal mutations - low probability
• acquired
– transfer of extra chromosomal DNA from a
resistant species to a sensitive one
– Plasmids
– Transposons
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Plasmids
– resistance factors or R factors transfered by
conjugation, transformation or transduction
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Transposons
– sequences that can move from
– plasmid >> chromosome
– plasmid>> plasmid
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Clinical Trials
• patient - has a diagnosed infection - two
possibilities:
– a) the new drug is the drug of choice by testing
– b) the patient has not responded to other drugs and
the new drug is testing well in the lab
• samples of blood etc. taken to determine all the
possible parameters:
– level of antimicrobial and presence of agent
– cultures of infecting agent taken 2 times per day
• disappearance of the bacteria and patient
recovery conclude a successful trial
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Minimum Inhibitory Concentration
• 1. test for antimicrobial activity
• 2. dilute antibiotic (pictures of tubes)
• 3. range selected obtained from therapeutic
index
• 4. add to medium
• 5. add pure culture of isolated bacteria
• 6. incubate - tubes that are clear after 16 hours
incubation at 35° C are subcultured
Reference: lab manual p. 270
– 0.1 ml removed and plated on suitably
rich medium usually the agar version of the liquid growth medium
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Kirby-Bauer Plate Sensitivity
• disks impregnated with various
concentrations of appropriate antibiotics are
placed aseptically on innoculated plates
• measurement of drug concentrations in the
blood preclinical trials
• subjects receive varying dose levels and
intervals of dosage
• pretrials usually determine the route of
Reference: lab manual p. 270
entry - oral or parenteral (injected
subcutaneously, intramuscularly, etc.)
• pretrials determine the carrier substance 4/13/2015
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Subjects Tested for Antimicrobial
Levels
• blood, lymph, urine, feces tested for
effective levels depending on disease
• also of concern is rapid metabolism
(catabolism) of the drug and also rapid
excretion
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Disk Diffusion Tests
• diffusion of antibiotic from disk controlled
by agar concentration
• Zone of Inhibition
– controlled by diffusion rate
– level of sensitivity
• each antibiotic is unique
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