Chemotherapy of Bacterial Infections ~~~~~~~~ Antibiotics

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

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.

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.

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.

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 1941 - spores from mold on coats of scientists

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.

* 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.

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

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

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

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

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.

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

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.

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

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

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

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)

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

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

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

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 http://www.healthsci.tufts.edu/apua/ROAR/roarhome.htm

Implications of Resistance www.roar.apua.org

REVIEW

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

What are main targets of Antibiotics? REVIEW

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

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

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

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

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

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

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

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

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

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