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Antibiotics

• Hugh B. Fackrell

• Filename: antibiot.ppt

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Outline• 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• 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 AntibioticsMost 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

• inhibit cell wall synthesis

• inhibit nucleic acid synthesis

• inhibit protein synthesis

• interfere with cell membrane function

Sulfa Drugs

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Sulfa vs PABA

PABAPABA

NHNH22HOOCHOOC

SulfanilamidSulfanilamidee

NHNH22NHNH22SOSO22

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Structure of Sulfa Drugs

ProntosilProntosil

SulfisoxazoleSulfisoxazole

SulfanilamideSulfanilamide

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Folic Acid MetabolismPABA + pteridinePABA + pteridine

ThymidineThymidine

DNADNA

PurinesPurines

DNA, RNADNA, RNA

MethionineMethionine

tRNa, tRNa, ProteinsProteins

Pteridine Pteridine synthetasesynthetase

Dihydropteroic acidDihydropteroic acid

[GTP][GTP]

Dihydrofolic AcidDihydrofolic Acid

Dihydrofolate Dihydrofolate SynthetaseSynthetase

L- GlutamineL- Glutamine

Tetrahydrofolic AcidTetrahydrofolic Acid

DihydrofolatDihydrofolate e synthetasesynthetase

2 NADPH2 NADPH

2 2 NADP+NADP+

SulfonamidSulfonamidee

TrimethopriTrimethoprimm

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ThymidineThymidine

DNADNA

PurinesPurines

DNA, RNADNA, RNA

MethionineMethionine

tRNa, tRNa, ProteinsProteins

Folic Acid InhibitionPABA + pteridinePABA + pteridine

Dihydropteroic acidDihydropteroic acid

Dihydrofolic AcidDihydrofolic Acid

Tetrahydrofolic AcidTetrahydrofolic Acid

SulfonamidSulfonamidee

TrimethopriTrimethoprimm

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Antibiotic Synergism

SulfisoxazoleSulfisoxazole

TrimethopriTrimethoprimm

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Antibiotic Synergism

• Sulfonamide + trimethoprim

• Effective dosage 10% of two separately

• Broader spectrum of action

• Reduce emergence of resistant strains

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

– 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– skin allergy

– anemia

– renal toxic

– hepato toxic

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Adverse Effects of Antibiotics

• Broad spectrum– Super infections – Candida albicans– Clostridium difficle– Staphylococcus– Gram -ve

TetraclycineTetraclycine– Depress bone Depress bone

marrowmarrow– ““Yellow teeth”Yellow teeth”

Pregnant womenPregnant women children <7 yearschildren <7 years

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Mode of Action of Antibiotics

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Mode of Action of Antibiotics• 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 NAM-pentapeptide 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 NAM-pentapeptide. 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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Murray 2.4 & 5.4, p. 10

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

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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

Membrane

Cytoplasm

Polymyxin action

• Polymyxin B binds to the cell membrane to disrupts its structural and permeability properties

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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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Reference: lab manual p. 270

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– 0.1 ml removed and plated on suitably rich medium -

usually the agar version of the liquid growth medium

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Reference: lab manual p. 270

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 entry - oral or parenteral (injected subcutaneously, intramuscularly, etc.)

• pretrials determine the carrier substance

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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