Concept of chemotherapy the MECHANISM OF ACTION and RESISTANCE

DEPARTMENT OF ALLIED HEALTH SCIENCEDEPARTMENT OF ALLIED HEALTH SCIENCEMSC IN BIOTECHNOLOGYMSC IN BIOTECHNOLOGY

MICROBIOLOGY PRESENTATIONCONCEPT OF CHEMOTHERAPYCONCEPT OF CHEMOTHERAPY

MECHANISM OF ACTION &RESISTANCEMECHANISM OF ACTION &RESISTANCEPRESENTED BY

“ESAM BASHIR YAHYA”

MAY intake 2015

PART I

CONCEPT OF Chemotherapy

Some important Definitions

• Chemotherapy The use of drugs to treat a disease.

• Antimicrobial drugs Interfere with the growth of microbes within a host.

• AntibioticSubstance produced by a microbe that, in small amounts, inhibits another microbe.

• Selective toxicity A drug that kills harmful microbes without damaging the host.

• Bacteriostatic/Modes of action that either kill or bacteriocidal inhibit growth.• Spectrum of activityRange of effect within or between groups of microbes; narrow vs. broad-spectrum.

The Development of Chemotherapy• Early 20th century

1904: Ehrlich found that the dye trypan red was effective against Trypanosoma (sleeping sickness)

Arspheniamine (Salvarsan) against syphilis.

Various dyes (including gentian violet); disinfectants; heavy metals were tried as antimicrobial chemotherapeutic agents.

Antisera were also used; for example, anti-sterptococcal antisera used against scarlet fever

Chemical agentsAgent Mechanism of

ActionUse

Peroxygens Oxidations Use in wounds and abrasions

Alcohols Protein denaturation

Skin disinfection

Organic acids Disrupt proteins- low pH

Used to control moleds

Detergents Disrupts cell membranes

Disinfection

Halogens derivative IodineChlorine

Inhibits protein action in bacteria

Surface area disinfection

The Development of Chemotherapy

• Sulfa drugs:

1927: Domagk discovered that the dye Prontosil Red was effective against staphlococcal and streptococcal infections; later in 1935 it was found that Protonsil red was converted to sulfonamide in the body

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

Origin of Antibiotics• These are agents that have been synthesized by bacteria or fungi against other organisms.

• Example – Penicillin was isolated from the mold Penicillium notatum. It was first observed by Alexander Fleming and later purified by Howard Florey.

• 1928 – Fleming discovered penicillin, produced by Penicillium.

• 1940 – Howard Florey and Ernst Chain performed first clinical trials of penicillin.

Figure 20.1

The Ideal Drug*1. Selective toxicity: against target pathogen but

not against host. LD50 (high) vs. MIC and/or MBC (low)

2. Bactericidal vs. bacteriostatic.3. Favorable pharmacokinetics:

reach target site in body with effective concentration4. Spectrum of activity: broad vs. narrow5. Lack of “side effects”

Therapeutic index: effective to toxic dose ratio6. Little resistance development

* There is no perfect drug* There is no perfect drug..

Features of Antimicrobial Drugs:

Selective Toxicity• Cause greater harm to microorganisms than to host.

• Chemotherapeutic index: lowest dose toxic to patient divided by dose. typically used for therapy.

Key Terms

• MIC = Minimal IInhibitorynhibitory Concentration. Lowest concentration of antimicrobial that inhibits growth of bacteria. Commonly used in clinical lab.

• MBC = Minimal Bactericidal Concentration. Concentration of an antimicrobial that kills bacteria. Used clinically only in special circumstances.

• Breakpoint = The MIC that is used to designate between susceptible and resistant. Arbitrarily set by a committee.

32 ug/ml16 ug/ml8 ug/ml4 ug/ml 2 ug/ml 1 ug/ml

Sub-culture to agar mediumMIC = 8 ug/mlMBC = 16 ug/ml

Minimal Inhibitory Concentration (MIC)vs.

Minimal Bactericidal Concentration (MBC)

Other Methods for Determining

Susceptibility

E-test®Kirby-Bauer

Disk Diffusion

Agar dilution

Concept of Breakpoint to Determine Susceptibility

Susceptibility Tests“Kirby-Bauer Disk-plate test”

Diffusion depends upon:1. Concentration2. Molecular weight3. Water solubility4. pH and ionization5. Binding to agar

Susceptibility Tests“Kirby-Bauer Disk-plate test”

Zones of Inhibition (~ antimicrobial activity) depend upon:

1. pH of environment2. Media components

Agar depth, nutrients3. Stability of drug4. Size of inoculum5. Length of incubation6. Metabolic activity of organisms

ANTIVIRAL DRUGS• Viruses pose a different set of problems for antibiotic therapy.They are obligate intracellular parasites.

Drugs that can eliminate the virus are dangerous to non-infected cells.

This makes selective toxicity difficult.• Many viruses difficult to grow.

It is difficult to test potential antiviral drugs.

ANTIVIRAL DRUGSMany acute viral infections have a short duration.

They are essentially over before antibiotics could be of any therapeutic use.

The lack of rapid tests means it is difficult to differentiate between various viral infections.

Successful antiviral drugs must eliminate all virions.

The escape of even one virion could restart the infectious cycle.

The first antibiotic to be used against viruses was the sulfa drug derivative thiosemicarbazone.

In 1960s amantadine developed for use against influenza.

ANTIVIRAL DRUGSAcyclovir is a specific and nontoxic drug: genital and oral herpes simplex infections & varicella-zoster (chickenpox and shingles).

Ganciclovir treat cytomegalovirus infection.

Foscarnet acts against DNA replication herpes infections.

Ribavirin Lassa fever infections. Amantadine - potent antiviral agent used against influenza A and not influenza B.

ANTIFUNGAL DRUGS• The emergence of diseases that render a host immunocompromised has led to increased secondary fungal infections.

• Drugs used for fungal infection have selective toxicity issues.Attacking common targets can cause serious side effects.

ANTIFUNGAL DRUGS• Polyenes are produced by the soil bacterium Streptomyces- used with caution because of side effects.

Azoles routinely used topically against athlete’s foot and vaginal yeast infection.

Ketoconazole is a broad spectrum derivative used for systemic fungal infections.

Griseofulvin produced by Penicillium, administered orally and is effective for superficial fungal infections.

Flucytosine interferes with DNA and RNA synthesis

DRUGS FOR PROTOZOA• The development of drugs for parasitic infections has lagged behind.

Parasitic infections do not occur often in developed nations.

There is no money in it.

• Two widely used anti-parasitic drugs are:QuinineMetronidazole.

DRUGS FOR HELMINTHS• Anti-helminthic drugs have also been largely ignored until recently.

Affected populations were not found in developed countries.

The popularity of sushi has led to an increase in tapeworm infestations.

Increased world travel has also increased helminth infections.

PART II

•Mechanisms of action of Antibacterial Drugs

Mechanisms of action of Antibacterial Drugs

1. Inhibit synthesis of essential metabolites.

2. Injury to plasma membrane.

3. Inhibit protein synthesis.

4. Inhibit nucleic acid synthesis.

5. Inhibit cell wall synthesis.

Antibiotic Mechanisms of Action

Transcription

Translation

Translation

Alteration of Cell Membrane Polymyxins Bacitracin Neomycin

Mechanism of Action1. ANTIMETABOLITE ACTION

Sulfonamides an analog of PABA, works by

competitive inhibition.

Trimethoprim-sulfamethoxazole a synergistic combination; useful

against UTIs.

Sulfonamides (Sulfa drugs)•Inhibit folic acid synthesis•Broad spectrum

Antibacterial Antibiotics Competitive Inhibitors

Figure 5.7

Mechanism of Action ANTIMETABOLITE ACTION

Mechanism of Action2. ALTERATION OF CELL MEMBRANES

Polymyxins and colistin destroys membranes active against gram negative

bacilli serious side effects used mostly for skin & eye

infections

Mechanism of Action ALTERATION OF CELL MEMBRANES

Mechanism of Action3. INHIBITION OF PROTEIN

SYNTHESIS:Steps in synthesis:1. Initiation2. Elongation3. Translocation4. Termination• Prokaryotes and eukaryotes, have a

different structure to ribosomes so can use antibiotics for selective toxicity against ribosomes of prokaryotes (70S) and eukaryotes (80S).

Mechanism of ActionINHIBITION OF PROTEIN SYNTHESIS

• 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 TobramycinAmikacin StreptomycinKanamycin SpectinomycinNeomycin


• Aminoglycosides • 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


• Macrolides: chloramphenicol & erythromycin bind to 50S subunit and blocks the translocation step

Mycoplasma

Legionella

S. pyogenes

Chloramphenicol: broad spectrum

Erythromycin:

Anaerobes

Typhoid

Meningitis


• 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

• 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


Mechanism of Action4. 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

Mechanism of Action INHIBITION OF DNA/RNA SYNTHESIS

Metronidazole breaks down into intermediate that causes breakage of DNA

active against:–protozoan infections–anaerobic gram negative infections Quinolones and fluoroquinolones

effect DNA gyrase broad spectrum

Mechanism of Action INHIBITION OF DNA/RNA SYNTHESIS

Mechanism of Action5. CELL WALL SYNTHESIS INHIBITORS

Steps in synthesis:1. NAM-peptide made in cytoplasm2. attached to bactoprenol in cell membrane3. NAG is added4. whole piece is added to growing cell wall5. crosslinks added

• the β-Lactams• the non β-Lactams

Bacterial Groups

Mechanism of Actionβ-Lactam Antibiotics

Penicillins Cephalosporins Carbapenems Monobactams

Mechanism of ActionCELL WALL SYNTHESIS INHIBITORS

β-Lactam ring structure


Action of β-Lactam antibiotics

1.Bactericidal; growing cells only

2.Drug links covalently to regulatory enzymes called PBPs (penicillin-binding proteins)

3.Blocks cross-linkage of peptidoglycan

Non - β-Lactams


Vancomycinactive 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 synthesisfor Mycobacterial infections

PART III

Resistance Physiological Mechanisms

Resistance to Antimicrobials

• Some microorganisms inherently resistant to effects of a particular drug.

• Other previously sensitive microorganisms can develop resistance through spontaneous mutations or acquisition of new genes (more later).

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

• Genetic spontaneous mutation of old

genes• Vertical evolution

Acquisition of new genes• Horizontal evolution

• Chromosomal Resistance• Extrachromosomal Resistance

• Plasmids, Transposons, Integrons

Plasmids• independent replicons

circular DNA.• Dispensable.• several genes:

drug resistance. metabolic enzymes. virulence factors.

• host range restricted or broad.

Plasmids• Size and types:-

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.

Agriculture

Inpatient

Outpatient

Antibiotic Use Leads to

Antibiotic Resistance

Reasons for Antibiotic Overuse :

Conclusions from 8 Focus GroupsPatient Concerns

• Want clear explanation

• Green nasal discharge

• Need to return to work

Physician Concerns• Patient expects antibiotic

• Diagnostic uncertainty

• Time pressure

Barden L.S. Clin Pediatr 1998;37:665Antibiotic Prescription

Unnecessary Antibiotics Cause

Resistance

Susceptible bacteria are killed

off.

A few hardy survivors are left

behind.

XXX

XX

X XX

The survivors can

withstand penicillin.

R

R

Jane takes penicillin.

• A variety of mutations can lead to antibiotic resistance.

• Mechanisms of antibiotic resistance1. Enzymatic destruction of drug.1. Enzymatic destruction of drug.2.2. Prevention of penetration of drug. Prevention of penetration of drug.3.3. Alteration of drug's target site. Alteration of drug's target site.4.4. Rapid ejection of the drug.Rapid ejection of the drug.

• Resistance genes are often on plasmids or transposons that can be transferred between bacteria.

Antibiotic Resistance

Mechanisms Of Antibiotic Resistance

• Bacteria are capable of becoming resistant through several mechanisms.

• One or many mechanisms may exist in an organism.

• Multidrug-resistant bacteria often have multiple mechanisms.

• Genes encoding resistance may exist on plasmid or chromosome.

Alteration in Target

Molecule

Decreased

Permeability

“Super Bugs”

Mechanisms of ResistanceAntibiotic Degrading Enzymes

• Sulfonation, phosphorylation, or esterifictation Especially a problem for aminoglycosides.

• β-lactamases Simple, extended spectrum β-lactamases (ESBL), cephalosporinases, carbapenemases.

Confer resistance to some, many, or all beta-lactam antibiotics.

May be encoded on chromosome or plasmid. More potent in gram-negative bacteria. Examples: S. aureus, H. influenzae, N. gonorrhoeae, E. coli,

Klebsiella sp., Enterobacter sp., Serratia sp., other enteric bacteria, anaerobes.

Extended Spectrum -lactamases

-lactamases capable of hydrolysing extended spectrum cephalosporins, penicillins, and aztreonam.

• Most often associated with E. coli and Klebsiella pneumoniae but spreading to other bacteria.

• Usually plasmid mediated.

• Aminoglycoside, ciprofloxacin and trimethoprim-sulfamethoxazole resistance often encoded on same plasmid.

• Has become a significant resistance determinate in acute and long-term care facility enteric pathogens.

Class A Carbapenemases• Most common in Klebsiella pneumoniae (KPC)

• Also seen in E. coli, Enterobacter, Citrobacter, Salmonella, Serratia, Pseudomonas and Proteus spp.

• Very often with multiple other drug resistance mechanisms, resistance profile similar to ESBL but also carbapenem resistant.

• Became problem in New York City first in 2002-2003 and is being increasingly recognized in Mid-Atlantic US.

• Spreading across species to other gram-negatives and enterobacteriaceae.

• Emerging in long-term care facilities

Mechanisms of Resistance

Decreased Permeability• Pseudomonas spp.• Affects many antibiotics including carbapenems.

Efflux Pumps• Pseudomonas spp. (multiple antibiotics).

• Tetracyclines.• Macrolides.

Mechanisms of Resistance

Mechanisms of ResistanceTarget Alteration

• Ribosome

• Tetracyclines

• Macrolides•S. pneumoniae, Staphylococcus sp., N. S. pneumoniae, Staphylococcus sp., N.

gonorrhoeae, enteric gram-negative gonorrhoeae, enteric gram-negative rods.rods.


1. Lack of entry – tet, fosfomycin2. Greater exit

efflux pumps tet (R factors)

3. Enzymatic inactivation bla (penase) – hydrolysis CAT – chloramphenicol acetyl transferase Aminogylcosides & transferases


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

Implicationsof Resistance

Is that the Is that the

End ??End ??Nope !!Nope !!

I don’t I don’t think sothink so

Thank youfor

listening

REFFERENCES REFFERENCES • http://en.wikipedia.org/wiki/Beta-lactamase

• http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=A1593|SIAL&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC

• http://abe.leeward.hawaii.edu/Protocols/QiagenSpinprepProtocol.htm

• http://www.openwetware.org/wiki/Brown_BIOL1220:Notebook/SynBio_in_Theory_and_Practice/Bacterial_Basics

• Microbiology, A clinical Approach -Danielle Moszyk-Strelkauskas-Garland Science 2010

http://en.wikipedia.org/wiki/Beta-lactamase

Concept of chemotherapy the MECHANISM OF ACTION and RESISTANCE

Documents