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Antimicrobial Therapy

Overview
If bacteria make it past our immune system and start reproducing inside our bodies, they cause disease. Certain bacteria produce chemicals that damage or disable parts of our bodies. Antibiotics work to kill bacteria.Antibiotics are specific to certain bacteria and disrupt their function.

What is an antibiotic?
Literal definition: Against (anti-) life (biotic) The more correct term is antimicrobial agent

What is an Antibiotic?
An antibiotic is a selective poison. It has been chosen so that it will kill the desired bacteria, but not the cells in your body. Each different type of antibiotic affects different bacteria in different ways. For example, an antibiotic might inhibit a bacteria's ability to turn glucose into energy, or the bacteria's ability to construct its cell wall. Therefore the bacteria dies instead of reproducing.

Sir Alexander Fleming


(1881 1955) One sometimes finds what one is not looking for

Penicillin

He observed inhibition of staphylococci on an agar plate contaminated by a Penicillium mold

Antibiotics Sources

Antibiotic-Groups: year of discovery or first treatment


1930 1940 1950 1960 1970 1980 1990 2000
1986 Oxazolidinone 1962 Chinolone 1962 Streptogramine 1962 Rifamycine 1958 Glykopeptide

1952 Makrolide
1950 Aminoglykoside 1949 Chloramphenicol 1949 Tetracycline 1940 Penicillin (Entdeckung 1928) 1936 Sulfonamide

Antimicrobial Therapy
Criteria that determine the effectiveness of antimicrobial agents selective destruction of the microorganism no or rare side effects on the host antimicrobial substances should have a specific action against molecules or enzymes or metabolism of the microorganism only

Disadvantages of Antimicrobial Therapy


1. Allergic reactions 2. Toxic effects on normal tissue 3. Disturbs host normal flora secondary infections

4. Development of resistant strains (producing enzymes that can destroy antibiotics; resistance genes e.g. in plasmids, transferred from to one bacterial cell to the other, mutation of the

target)

Antimicrobial Therapy
Natural antibiotic agents:
Metabolic products produced by microorganisms: Penicillium notatum penicillin Streptomyces spp. streptomycin

Semi-synthetic antibiotic agents:


chemically modified natural agents (large group of modern antibiotics)

synthetic antibiotic agents:


Chemically related to natural antibiotics but completely industrially manufactured

Classification of Antibiotics
Chemical Structure Spectrum of Activity Mechanism of Action

ANTIBIOTICS CLASSIFICATION
Very diverse class of compounds Generally grouped according chemical structures

Aminoglysides Cephalosporins and related beta lactams Glycopeptides Macrolides Penicillins Quinolones Sulphonamides and diaminopyrimidines Tetracyclines

Bacteriostatic vs. Bactericidal


bacteriostatic - stop growth (don't kill) bactericidal kill cells

bacteriostatic

bacteriocidal

log viable count

Antibiotics Modes of Action

bacteriostatic

bactericidal

How antibiotics work


Inhibition of nucleic acid synthesis
Rifampicin; Chloroquine

Inhibition of protein synthesis


Tetracyclines; Chloramphenicol

Action on cell membrane


Polyenes; Polymyxin

Interference with enzyme system


Sulphamethoxazole

Action on cell wall


Penicillin; Vancomycin
penicillin works by blocking the formation of peptide bonds in the bacterial cell wall and thereby weakens it, leaving the bacterium susceptible to osmotic lysis

Antibiotics and Chemotherapeutics Modes of Action


cell-wall synthesis DNA-replication DNA-dependent RNA polymerase

cell-wall
protein-synthesis

folic acid metabolism

protein-synthesis

cell-membrane
bacterial cell

Antibiotic Modes of Action

Drug Vancomycin

Mode of Action Binds to D-Ala-D-Ala terminus; inhibits transpeptidation enzymes of cell wall

Cephalosporins Inhibits transpeptidation enzymes. Activates lyctic


Carbenicillin Rifampin Ciprofloxacin Tetracyclines Streptomycin Sulfonamides Polymyxin B Inhibits transpeptidation enzymes. Activates lyctic enzymes of cell wall Blocks RNA synthesis: binds to, inhibits DNAdependent RNA polymerase Inhibits bacterial DNA gyrase; interferes with DNAinvolved activities like DNA replication Interfere with aminoacyl-tRNA binding: binds to 30S ribosomal subunit Causes misreading of mRNA: binds to 30S ribosomal subunit Interferes with synthesis of folic acid by competition with p-aminobenzoic acid Disrupts structure and permeability of plasma mambrane by binding with it

from: http://project.bio.iastate.edu/Courses/MIPM302/302new/9_1chemother.html

Inhibitors of Cell Wall Synthesis

Beta-Lactam Antibiotics
Types of -Lactam Antibiotics Penicillins Examples Penicillin Amoxicillin Dicloxacillin Cephalexin, cefazolin (1st generation) Cefuroxime (2nd generation) Ceftriaxone, ceftazidime (3rd generation) Cefepime (4th generation) Ertapenem Imipenem Meropenem Aztreonam Amoxicillin-clavulinate Ampicillin-sulbactam Piperacillin-tazobactam

Cephalosporins

Carbapenems

Monobactams Beta-lactamase inhibitors

Penicllins
Derived from the fungus Penicillium Therapeutic concentration in most tissues Poor CSF penetration Renal excretion Side effects: hypersensitivity, nephritis, neruotoxicity, platelet dysfunction

Cephalosporins
Structurally similar to penicillins Therapeutic concentration in many tissues, 3rd and 4th generation into CSF Renal Excretion Side Effects
allergy disulfiram-like effect anti-Vitamin K

Generations of Cephalosporins

Monobactams
Aztreonam
single beta lactam ring narrow spectrum: gramnegative aerobes
Enterobacteriacea Pseudomonas

given IV/IM renal excretion little cross-reactivity with other beta lactams side effects: phlebitis, rash, elevated LFTs

Carbapenems
Meropenem/Imipenem
broad spectrum active against MRSA given IV penetrates CSF renal metabolism and excretion addition of cilastin side effects: GI upset, eosinophilia, neutropenia, lowering of seizure threshold

Vancomycin
Tricyclic glycopeptide Inhibits synthesis of phospholipids and cross-linking of peptidoglycans Activity against gram-positive organisms Useful for beta lactam resistant infections Widely distributed, penetrates CSF Renal elimination, follows creatinine cl. Side effects: phlebitis, red man syndrome, ototoxicity, nephrotoxicity

Protein Synthesis Inhibitors


Human Ribosome
80S
40S 60S

Bacterial Ribosome
70S
30S 50S

Tetracyclines
Isolated from Streptomyces aureofaciens Reversibly bind 30S ribosomal subunit Penetrate sinus mucosa, saliva and tears Metabolized in liver-->excreted in bile--> reabsorbed-->eliminated in urine Side effects: GI upset, hepatotoxicity, photosensitivity, bony deposition Contraindicated in pregnant or breast feeding women, children under 8 y/o

Aminoglycosides
Derived from Streptomyces and Micormonospora Irreversible binding to 30S subunit Actively transported into bacterial cells Variable tissue penetration, unreliable CSF levels Concentrate within perilymph Renal elimination Nephrotoxicity, ototoxicity, neurotoxicity

Aminoglycosides

Macrolides
Macrocyclic lactone structure Irreversible binding to 50S subunit Therapeutic concentrations in oropharyngeal and respiratory secretions No CSF penetration Metabolized in liver, excreted in feces and urine Side effects: GI upset, ototoxicity, hepatotoxicity

Erythromycin

Alternate Macrolides
Clarithromycin Azithromycin

Chloramphenicol
Isolated from Streptomyces Reversible binding to 50S subunit Broad spectrum of activity Indicated for severe anaerobic infections or unresponsive life-threatening infections Widely distributed, enters CSF Metabolized in liver (inhibits P-450), eliminated in urine Toxicities: reversible anemia, hemolytic anemia, aplastic anemia, gray baby syndrome

Clindamycin
Semisynthetic derivative of Lincomycin Irreversible binding to 50S subunit Covers anaerobes and gram + aerobes Widely useful for head and neck infections Penetrates saliva, sputum, pleural fluid, and bone, but not CSF Metabolized in liver-->reabsorbed-->eliminated in urine Side effects: rash, neutropenia/thrombocytopenia, pseudomembranous colitis

Inhibitors of Metabolism
Sulfonamides Trimethoprim Interfere with the production of folic acid coenzymes that are required for purine and pyrimidine synthesis

Sulfonamides
Derived from prontosil Competitve antagonist of PABA Wide distribution, penetrate CSF, cross placenta Metabolized in liver, eliminated in urine Side effects: rash, angioedema, StevensJohnson syndrome, kernicterus Avoid in pregnancy and infants

Trimethoprim
Inhibits dihydrofolate reductase 1000x higher affinity for bacterial enzyme than human enzyme Similar spectrum and pharmacokinetic profile as sulfas Side effects: folate deficiency anemia, leukopenia, granulocytopenia

Co-Trimoxazole (TMP/SMX)
Combination gives synergistic antibacterial action

Inhibitors of Nucleic Acid Function/Synthesis


Fluoroquinolones
Bind bacteria DNA gyrase (topoisomerase II) Concentrate in sinus and middle ear mucosa, penetrate cartilage and bone Partially metabolized in liver-->GI or renal excretion Side effects: nausea, dizziness, phototoxicity, nephrotoxicity Avoid in pregnant or nursing women ? Use in children--possible effect on articular cartilage

Fluoroquinolone Antibiotics
Fluoroquinolone Antibiotic Ciprofloxacin Spectrum of Activity Many Gram negative bacteria Some Pseudomonas Atypicals (Legionella, Mycoplasma) Mycobacteria Many Gram negative bacteria Some Pseudomonas Many Gram positives, including Strep Atypicals (Legionella, Mycoplasma) Mycobacteria Many Gram negative bacteria Many Gram positives, including Strep Anaerobes Atypicals (Legionella, Mycoplasma) Mycobacteria

Levofloxacin

Moxifloxacin

Fluoroquinolones

Antibiotic Resistance
innate (natural) resistance primary resistance, is based on a genetically caused insensitivity of a type of bacteria to a certain antibiotic e.g. inefficacy of penicillin to Ps. aeruginosa acquired resistance

secondary resistance, after or during antibiotic treatment, activation of enzymes or genes

Avoid disadvantages by:


avoiding indiscriminate use of antibiotics maintain proper levels for sufficient (prescribed) length of time use a combination of 2 or more antibiotics in severe infections to prevent resistance synergistic effects of the antibiotics perform antibiotic sensitivity test to determine the most effective antibiotics

Mechanisms of Antibiotic Resistance

Grace Yim and Fan Sozzi

Mechanisms of Resistance
1) Accumulation barriers to an antimicrobic due to impermeability or active efflux 2) Alterations of an antimicrobic target which render it insusceptible 3) Inactivation of an antimicrobic by an enzyme produced by the microorganism

Mechanisms of Antibiotic Resistance


Antibiotic Chloramphenicol Tetracycline -lactams, Erythromycin, Lincomycin Method of resistance reduced uptake into cell active efflux from the cell eliminates or reduces binding of antibiotic to target

-lactams, Erythromycin
Aminoglycosides, Chloramphenicol, Fosfomycin, Lincomycin -lactams, Fusidic Acid Sulfonamides, Trimethoprim Sulfonamides, Trimethoprim

hydrolysis
inactivation of antibiotic by enzymatic modification sequestering of the antibiotic by protein binding metabolic bypass of inhibited reaction overproduction of antibiotic target (titration)

Bleomycin

binding of specific immunity protein to antibiotic

Influx/Efflux
Bacteria such as streptococci, enterococci, and anaerobes lack the necessary oxidative pathways for transport of aminoglycosides. Some bacteria have energy-dependent efflux mechanisms that pump either tetracyclines or fluoroquinolones from the cell

Altered Target
Antimicrobics act by binding and inactivating their target, which is typically a crucial enzyme or ribosomal site. Substitutions of one amino acid in a protein can alter its binding.
First generation aminoglycosides and quinolones only bind one site. Newer agents bind at multiple sites on their target making resistance improbable.

An important example : -lactam family In widely divergent gram-pos and gramneg species changes in one or more of peptidoglycan transpeptidase penicillinbinding proteins (PBP) have been correlated with decreased susceptibility to multiple -lactams Causes: point mutations, substitutions of amino acid sequence, and synthesis of a new enzyme.

Enzymatic Inactivation Most powerful and robust of resistance mechanisms -Lactamases - 100s of enzymes with variable activity against - lactam substrates Typically demonstrate high-level resistance with MICs above therapeutic range

How Antibiotic Resistance Happens

Horizontal Gene Transfer


( Grace Yim and Fan Sozzi)

Antibiotic Sensitivity Test


Measurement of the sensitivity of microorganisms to antibiotics and chemotherapeutics is of great importance in the rational use of chemotherapy Methods a) agar diffusion - Kirby-Bauer test (= disc test)

- Etest (= strip test)


b) broth dilution

Antibiotic Sensitivity Test


Kirby-Bauer Test (disc diffusion test)

Antibiotic Sensitivity Test


Etest (strip test)

Antibiotic Sensitivity Test Broth Dilution Method


antibiotic (dilution series) + bacterial suspension (standard amount) growth ?

MIC minimal inhibitory concentration

The MIC is the lowest concentration that prevents ___________ MIC>1.6 mg/L This value is compared with the ___________ for that antibiotic The test organism is then classified as susceptible, intermediate or resistant

Minimal inhibitory concentrations broth

Minimal inhibitory concentrations - agar


Performed in a similar manner to the broth test Two-fold serial dilution of ____________ are prepared in Mueller Hinton agar and poured into plates (standard depth) A _____________ inoculum is spotted onto the agar plate, along with susceptible and resistant control bacteria The plates are incubated for 18-24 hours at 35oC The __________ is the lowest antibiotic concentration that completely prevents growth

Minimal inhibitory concentrations - agar


Growth control (no antibiotic)
18 strains grow

Antimicrobial concentration 4mg/L


Only 7/18 strains grow

Antimicrobial concentration 8mg/L


Only 5/18 strains grow The MIC for this organism is >8mg/L

E-Test strips
E-Tests are 5 x 0.5 cm plastic strips
On the bottom is a gradient of stabilized antibiotic On the top is an exponential MIC reading scale (mg/L)

An agar plate is inoculated with a standard amount of test organism and the strip placed on top After incubation, the 'MIC' is read where the zone of inhibition intercepts the strip The advantage of this method is speed and different growth media can be used, allowing for the growth of fastidious bacteria

Automated systems Vitek 2


Playing card sized format, 64 wells Test up to 20 different antimicrobial agents at one time System is completely automated bacteria in one end, data out the other Results can be obtained faster than traditional methods
Uses a more sensitive fluorescent reader Takes readings every 15 minutes

The big disadvantage is initial cost

Common Antibiotics for Gram Positive Infections


Gram Positive Bacterium Group A Streptococcus Enterococcus Ampicillin-resistant enterococcus Vancomycin-resistant enterococcus Methicillin-susceptible Staph aureus Methicillin-resistant Staph aureus Coagulase-negative staphylococci Commonly Used Antibiotics Penicillin Amoxicillin Ampicillin Amoxicillin Vancomycin Linezolid Daptomycin Nafcillin Cefazolin Vancomycin Daptomycin Vancomycin Daptomycin

ANTITUBERCULOSIS CLASSIFICATION
Miscellaneous group of antibacterials Include

Cycloserine Ethambutol Isoniazid Pyrazinamide Rifampicin

Antifungal Agents
Azoles Fluconazole Ketoconazole Itraconazole Posaconazole Voriconazole
Echinocandins Anidulafungin Caspofungin Micafungin Polyenes Amphotericin B Lipid formulations of Amphotericin B

Antiviral Agents
Virus Herpes simplex virus Antiviral Agents Acyclovir Famciclovir Valacyclovir

Varicella-Zoster virus

Acyclovir Famciclovir Valacyclovir


Ganciclovir Valganciclovir Foscarnet Cidofovir Amantadine Rimantadine Oseltamivir Zanamivir

Cytomegalovirus

Influenza virus

Current Antiretroviral Agents


Nucleoside/tide RTIs zidovudine, AZT didanosine, ddI zalcitabine, ddC stavudine, d4T lamivudine, 3TC abacavir, ABC tenofovir, TDF emtricitabine, FTC Non-nucleoside RTIs
nevirapine, NVP delavirdine, DLV efavirenz, EFV etravirine, ETR

Fixed Dose Combos ZDV/3TC ZDV/3TC/ABC ABC/3TC TDF/FTC TDF/FTC/EFV Entry Inhibitors enfuvirtide, T-20 maraviroc, MVC Integrase Inhibitors raltegravir, RAL

Protease Inhibitors saquinavir, SQV ritonavir, RTV indinavir, IDV nelfinavir, NFV amprenavir, APV lopinavir/r, LPV/r atazanavir, ATV fosamprenavir, FPV tipranavir, TPV darunavir, DRV

Thank you for not asking questions

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