1 applied microbiology 9 th lecture: chapter 12. 2 the elements of chemotherapy principles of...

11

Applied MicrobiologyApplied Microbiology

99thth lecture: Chapter 12 lecture: Chapter 12

22

The Elements of ChemotherapyThe Elements of Chemotherapy

Principles of Antimicrobial TherapyPrinciples of Antimicrobial Therapy

44



Origins of Antibiotic DrugsOrigins of Antibiotic Drugs

Alexander Fleming 1928 discovery of Penicillin

55



Origins of Antibiotic DrugsOrigins of Antibiotic Drugs

AntibioticsAntibiotics are common are common metabolic productsmetabolic products of of aerobic aerobic spore-forming bacteria and fungi.spore-forming bacteria and fungi.• bacteria in genera bacteria in genera StreptomycesStreptomyces andand BacillusBacillus• molds in genera molds in genera PenicilliumPenicillium and and CephalosporiumCephalosporium

By inhibiting the other microbes in the same habitat, By inhibiting the other microbes in the same habitat, antibiotic producers have less competition for nutrients antibiotic producers have less competition for nutrients and space.and space.

Streptomyces

66



77


Interactions between Drug and MicrobeInteractions between Drug and Microbe

Antimicrobial drugs should be Antimicrobial drugs should be selectively toxicselectively toxic - drugs - drugs should kill or inhibit microbial cells without simultaneously should kill or inhibit microbial cells without simultaneously damaging host tissues.damaging host tissues.

As the characteristics of the infectious agent become As the characteristics of the infectious agent become more similar to the vertebrate host cell, complete more similar to the vertebrate host cell, complete selective toxicityselective toxicity becomes becomes more difficult to achievemore difficult to achieve and and more side effectsmore side effects are seen. are seen.

88


Interactions between Drug and MicrobeInteractions between Drug and MicrobeMechanisms of Drug Mechanisms of Drug

ActionAction

1010


Interactions between Drug and MicrobeInteractions between Drug and MicrobeMechanisms of Drug Mechanisms of Drug

ActionAction

1111



Mechanisms of Drug Action – Spectrum of DrugMechanisms of Drug Action – Spectrum of Drug

Spectrum – range of activity of a drugSpectrum – range of activity of a drug• narrow-spectrumnarrow-spectrum – effective on a small range of – effective on a small range of

microbesmicrobes target a target a specific cell componentspecific cell component that is found only that is found only

in certain microbesin certain microbes

• broad-spectrumbroad-spectrum – greatest range of activity – greatest range of activity target cell components common to most target cell components common to most

pathogenspathogens

1212



Mechanisms of Drug Action – Cell Wall SynthesisMechanisms of Drug Action – Cell Wall Synthesis

Most bacterial cell walls contain Most bacterial cell walls contain peptidoglycan.peptidoglycan. PenicillinsPenicillins and and cephalosporinscephalosporins blockblock synthesis of synthesis of

peptidoglycanpeptidoglycan, causing the cell wall to lyse., causing the cell wall to lyse. Active on young, growing cellsActive on young, growing cells PenicillinsPenicillins do not penetrate the outer membrane and are do not penetrate the outer membrane and are

less effective against Gram-negative bacteria.less effective against Gram-negative bacteria. Broad spectrum penicillins Broad spectrum penicillins and and cephalosporinscephalosporins can can cross cross

the cell walls the cell walls ofof Gram-negative bacteria. Gram-negative bacteria.

1313




Healthy cell

Drug affected cell

1414




Penicillin and Cephalosporin block peptidase to link NAMs

1515


Interactions between Drug and MicrobeInteractions between Drug and MicrobeMechanisms of Drug Action – Disruption of Membrane Mechanisms of Drug Action – Disruption of Membrane

FunctionFunction

A cell with a A cell with a damaged membranedamaged membrane dies from disruption in dies from disruption in metabolism or lysis.metabolism or lysis.

These drugs have These drugs have specificityspecificity for a particular microbial for a particular microbial group, based on differences in types of lipids in their cell group, based on differences in types of lipids in their cell membranes.membranes.

Polymyxins Polymyxins interact with interact with phospholipids and cause leakagephospholipids and cause leakage, , particularly in Gram-negative bacteria.particularly in Gram-negative bacteria.

Amphotericin BAmphotericin B and and nystatinnystatin form form complexes with sterolscomplexes with sterols on fungal membranes which causes leakage.on fungal membranes which causes leakage.

-> Antimicrobial Peptides !!!-> Antimicrobial Peptides !!!

1616


Interactions between Drug and MicrobeInteractions between Drug and MicrobeMechanisms of Drug Action – Disruption of Membrane Mechanisms of Drug Action – Disruption of Membrane

FunctionFunction

1717


Interactions between Drug and MicrobeInteractions between Drug and MicrobeMechanisms of Drug Action – Inhibition of DNA and RNA Mechanisms of Drug Action – Inhibition of DNA and RNA

SynthesisSynthesis

May block synthesis of nucleotides, May block synthesis of nucleotides, inhibit replicationinhibit replication, or , or stop transcriptionstop transcription

ChloroquineChloroquine binds and cross-links the double helix; binds and cross-links the double helix; quinolonesquinolones inhibit DNA helicases (Replication). inhibit DNA helicases (Replication).

Antiviral drugsAntiviral drugs that are analogs of purines and pyrimidines that are analogs of purines and pyrimidines insert in viral nucleic acid, insert in viral nucleic acid, preventing replicationpreventing replication..

1818



SynthesisSynthesis

DNA Synthesis:

Cell devision-> DNA Replication

1919



SynthesisSynthesis

DNA Replication

One strand template for new strand -> Replication

2020



SynthesisSynthesis

RNA Synthesis -> Transcription

mRNA molecule made from DNA

2121



Mechanisms of Drug Action – Inhibition of Protein SynthesisMechanisms of Drug Action – Inhibition of Protein Synthesis

RibosomesRibosomes of eukaryotes differ in size and structure from of eukaryotes differ in size and structure from prokaryotes; antimicrobics usually have a prokaryotes; antimicrobics usually have a selective action selective action against prokaryotesagainst prokaryotes; can also damage the eukaryotic ; can also damage the eukaryotic mitochondriamitochondria

AminoglycosidesAminoglycosides (streptomycin, gentamycin) insert on sites (streptomycin, gentamycin) insert on sites on the 30S subunit and cause on the 30S subunit and cause misreading of mRNAmisreading of mRNA..

TetracyclinesTetracyclines block attachment of tRNA on the A acceptor block attachment of tRNA on the A acceptor site and site and stop further synthesisstop further synthesis..

2222



Mechanisms of Drug Action – Inhibition of Protein SynthesisMechanisms of Drug Action – Inhibition of Protein Synthesis

Protein Synthesis -> Translation

mRNA molecule used to make protein

2323



Mechanisms of Drug Action – Inhibition of PathwaysMechanisms of Drug Action – Inhibition of Pathways

SulfonamidesSulfonamides and and trimethoprimtrimethoprim block enzymes required block enzymes required for tetrahydrofolate synthesis needed for for tetrahydrofolate synthesis needed for DNA and RNA DNA and RNA synthesis.synthesis.

Competitive inhibitionCompetitive inhibition – drug competes with normal – drug competes with normal substrate for enzyme’s active sitesubstrate for enzyme’s active site

Synergistic effectSynergistic effect – an – an additive effectadditive effect, achieved by , achieved by multiple drugs working together, requiring a lower dose multiple drugs working together, requiring a lower dose of eachof each

2424

Interactions between Interactions between Drug and MicrobeDrug and Microbe

Mechanisms of Drug Action – Mechanisms of Drug Action – Inhibition of PathwaysInhibition of Pathways

Competitive Inhibition of Sulfonamides

2525


Survey of Major Antimicrobial Drug GroupsSurvey of Major Antimicrobial Drug Groups

Antibacterial drugsAntibacterial drugs• antibioticsantibiotics• synthetic drugssynthetic drugs

Antifungal drugsAntifungal drugs

Antiprotozoan drugsAntiprotozoan drugs

Antiviral drugsAntiviral drugs

-> About 260 different antimicrobial drugs are -> About 260 different antimicrobial drugs are classified in 20 drug families.classified in 20 drug families.

2626



Antibacterial drugs acting on cell wall –> β-lactam antibiotics

Large Large diverse groupdiverse group of compounds of compounds Could be Could be synthesizedsynthesized in the laboratory in the laboratory More economical to obtain natural penicillin through More economical to obtain natural penicillin through

microbial fermentationmicrobial fermentation and and modify it to semi-syntheticmodify it to semi-synthetic formsforms

PenicilliumPenicillium chrysogenumchrysogenum – major source – major source All consist of 3 parts:All consist of 3 parts:

• thiazolidine ringthiazolidine ring• beta-lactam ringbeta-lactam ring• variable side chain dictating microbial activityvariable side chain dictating microbial activity

2727



Antibacterial drugs acting on cell wall –> β-lactam antibiotics

Beta-lactam antimicrobials - all contain a highly reactive Beta-lactam antimicrobials - all contain a highly reactive 3 carbon, 1 nitrogen ring -> 3 carbon, 1 nitrogen ring -> ββ--lactam ringlactam ring

Primary mode of action is to Primary mode of action is to interfere with cell wall interfere with cell wall synthesis.synthesis.

Greater than ½ of all antimicrobic drugs are beta-Greater than ½ of all antimicrobic drugs are beta-lactams.lactams.

Penicillins and cephalosporins most prominent beta-Penicillins and cephalosporins most prominent beta-lactams.lactams.

2828



Antibacterial drugs acting on cell wall –> β-lactam antibiotics (Penicillins)

2929




3030




Penicillins G and V most important natural formsPenicillins G and V most important natural forms Penicillin is the drug of choice for Penicillin is the drug of choice for Gram-positive cocciGram-positive cocci

(streptococci) and (streptococci) and some Gram-negative bacteriasome Gram-negative bacteria (meningococci and syphilis spirochete).(meningococci and syphilis spirochete).

Semisynthetic penicillinsSemisynthetic penicillins – ampicillin, carbenicillin and – ampicillin, carbenicillin and amoxicillin have amoxicillin have broader spectrabroader spectra – Gram-negative – Gram-negative enteric rodsenteric rods

Penicillinase-resistantPenicillinase-resistant – methicillin, nafcillin, cloxacillin – methicillin, nafcillin, cloxacillin Primary problems – Primary problems – allergiesallergies and and resistant strainsresistant strains of of

bacteriabacteria

3131



Antibacterial drugs acting on cell wall –> β-lactam antibiotics (Cephalorins)

Account for majority of all antibiotics administeredAccount for majority of all antibiotics administered Isolated from Isolated from Cephalosporium acremoniumCephalosporium acremonium mold mold Synthetically Synthetically altered beta-lactamaltered beta-lactam structure structure Relatively broad-spectrumRelatively broad-spectrum, resistant to most , resistant to most

penicillinases, & cause penicillinases, & cause fewer allergic reactionsfewer allergic reactions Some are given orally; many must be administered Some are given orally; many must be administered

parenterally.parenterally. Generic names have root – Generic names have root – cef, ceph, cef, ceph, or or kef.kef.

3232




4 generations exist4 generations exist: each group : each group more effective against more effective against Gram-negativesGram-negatives than the one before with improved dosing than the one before with improved dosing schedule and fewer side effectsschedule and fewer side effects

• first generationfirst generation – cephalothin, cefazolin – most effective against – cephalothin, cefazolin – most effective against Gram-positive cocci and few Gram-negativeGram-positive cocci and few Gram-negative

• second generationsecond generation – cefaclor, cefonacid – – cefaclor, cefonacid – more effective more effective against Gram-negative bacteriaagainst Gram-negative bacteria

• third generationthird generation – cephalexin, ceftriaxone – – cephalexin, ceftriaxone – broad-spectrum broad-spectrum activityactivity against enteric against enteric bacteria with beta-lactamasesbacteria with beta-lactamases

• fourth generationfourth generation – cefepime – – cefepime – widest rangewidest range; both Gram- ; both Gram- negative and Gram-positivenegative and Gram-positive

3333




3434



Antibacterial drugs acting on cell wall –> non β-lactam antibiotics

vancomycin vancomycin – – narrow-spectrumnarrow-spectrum, most effective in treatment , most effective in treatment of of Staphylococcal infectionsStaphylococcal infections in cases of penicillin and in cases of penicillin and methicillin resistance or if patient is allergic to penicillin; methicillin resistance or if patient is allergic to penicillin; toxic and hard to administertoxic and hard to administer; restricted use; restricted use

bacitracin bacitracin – – narrow-spectrumnarrow-spectrum produced by a strain of produced by a strain of Bacillus Bacillus subtilissubtilis; used topically in ointment; used topically in ointment

isoniazidisoniazid (INH) – works by interfering with mycolic acid (INH) – works by interfering with mycolic acid synthesis; used to treat infections with synthesis; used to treat infections with Mycobacterium Mycobacterium tuberculosistuberculosis; ; oral doses in combination with other oral doses in combination with other antimicrobials such as rifampin, ethambutolantimicrobials such as rifampin, ethambutol

3535



Antibacterial drugs acting on Protein Synthesis - Aminoglycosides

Aminoglycosides Aminoglycosides – composed of 2 or – composed of 2 or more amino sugars and an aminocyclitol more amino sugars and an aminocyclitol (6C) ring; binds (6C) ring; binds ribosomal subunitribosomal subunit

Products of various species of soil Products of various species of soil actinomycetesactinomycetes in genera in genera StreptomycesStreptomyces and and MicromonosporaMicromonospora

Broad-spectrumBroad-spectrum, , inhibit protein inhibit protein synthesissynthesis, especially useful against , especially useful against aerobic aerobic Gram-negative rodsGram-negative rods and certain and certain gram-positive gram-positive bacteriabacteria• streptomycin – bubonic plague, tularemia, TBstreptomycin – bubonic plague, tularemia, TB• gentamicin – less toxic, used against Gram-gentamicin – less toxic, used against Gram-

negative rodsnegative rods• newer – tobramycin and amikacin Gram-newer – tobramycin and amikacin Gram-

negative bacterianegative bacteriaStreptomycin

3636



Antibacterial drugs acting on Protein Synthesis - Tetracyclines

Broad-spectrumBroad-spectrum, block protein synthesis by , block protein synthesis by binding binding ribosomesribosomes

Aureomycin, terramycin, tetracycline, doxycycline and Aureomycin, terramycin, tetracycline, doxycycline and minocycline – minocycline – low cost oral drugslow cost oral drugs; ; side effectsside effects are a are a concernconcern

Treatment for STDs, Rocky Mountain spotted fever, Treatment for STDs, Rocky Mountain spotted fever, Lyme disease, typhus, acne and protozoaLyme disease, typhus, acne and protozoa

3737



Antibacterial drugs acting on Protein Synthesis - Chloramphenicol

Isolated from Isolated from StreptomycesStreptomyces venezuelae; venezuelae; no longer no longer derived from natural sourcederived from natural source

Potent Potent broad-spectrum broad-spectrum drug with unique nitrobenzene drug with unique nitrobenzene structurestructure

Blocks peptide bondBlocks peptide bond formation formation Very toxicVery toxic, restricted uses, can cause irreversible , restricted uses, can cause irreversible

damage to bone marrowdamage to bone marrow Typhoid fever, brain abscesses, rickettsial and Typhoid fever, brain abscesses, rickettsial and

chlamydial infectionschlamydial infections

3838



Antibacterial drugs acting on Protein Synthesis - Macrolides

ErythromycinErythromycin –large lactone ring with sugars; attaches to –large lactone ring with sugars; attaches to ribosomal 50s subunitribosomal 50s subunit

Broad-spectrumBroad-spectrum, fairly low toxicity, fairly low toxicity Taken orally for Mycoplasma pneumonia, legionellosis, Taken orally for Mycoplasma pneumonia, legionellosis,

Chlamydia, pertussis, diphtheria and as a prophylactic Chlamydia, pertussis, diphtheria and as a prophylactic prior to intestinal surgeryprior to intestinal surgery

For penicillin-resistant – gonococci, syphilis, acneFor penicillin-resistant – gonococci, syphilis, acne Newer semi-synthetic macrolides – clarithomycin, Newer semi-synthetic macrolides – clarithomycin,

azithromycinazithromycin

3939



Antibacterial drugs blocking metabolic pathways

Most are Most are syntheticsynthetic; most ; most important are important are sulfonamidessulfonamides, or , or sulfa drugs - first antimicrobic sulfa drugs - first antimicrobic drugsdrugs

Narrow-spectrumNarrow-spectrum; block the ; block the synthesis of folic acidsynthesis of folic acid by by bacteriabacteria• sulfisoxazole – shigellosis, UTI, sulfisoxazole – shigellosis, UTI,

protozoan infectionsprotozoan infections• silver sulfadiazine –burns, eye silver sulfadiazine –burns, eye

infectionsinfections• trimethoprim – given in combination trimethoprim – given in combination

with sulfamethoxazole – UTI, PCPwith sulfamethoxazole – UTI, PCP

4040



Antifungal drugs Fungal cells are eukaryotic; a drug that is toxic to fungal cells also Fungal cells are eukaryotic; a drug that is toxic to fungal cells also

toxic to human cells -> toxic to human cells -> more problematicmore problematic Three examples out of five antifungal drug groups:Three examples out of five antifungal drug groups:

• macrolide polyene - macrolide polyene - amphotericin B, nystatinamphotericin B, nystatin• synthetic azolessynthetic azoles – broad-spectrum; ketoconazole, clotrimazole, – broad-spectrum; ketoconazole, clotrimazole,

miconazolemiconazole• flucytosineflucytosine – analog of cytosine; cutaneous mycoses or in – analog of cytosine; cutaneous mycoses or in

combination with amphotericin B for systemic mycosescombination with amphotericin B for systemic mycoses

PolyenesAzole Flucytosine

4141



Antiviral drugs

Selective toxicity is almost impossibleSelective toxicity is almost impossible due to obligate due to obligate intracellular parasitic nature of viruses.intracellular parasitic nature of viruses.

Block penetrationBlock penetration into host cell into host cell Block transcription or translationBlock transcription or translation of viral genetic material of viral genetic material

• nucleotide analogs nucleotide analogs acyclovir – herpesvirusesacyclovir – herpesviruses ribavirin- a guanine analog – RSV, hemorrhagic ribavirin- a guanine analog – RSV, hemorrhagic

feversfevers AZT – thymine analog - HIVAZT – thymine analog - HIV

Prevent maturationPrevent maturation of viral particles of viral particles• protease inhibitors – HIVprotease inhibitors – HIV

4343



Antiviral drugs – Drugs for treating HIV infection and AIDS

Retrovirus offers Retrovirus offers 2 targets for chemotherapy2 targets for chemotherapy::• interference with interference with viral DNA synthesisviral DNA synthesis from viral RNA from viral RNA

using nucleoside using nucleoside reverse transcriptase inhibitorsreverse transcriptase inhibitors (nucleotide analogs)(nucleotide analogs)

• interference with interference with synthesis of DNAsynthesis of DNA using nonnucleoside using nonnucleoside reverse transcriptase inhibitorsreverse transcriptase inhibitors

-> azidothymidine (AZT) – thymine analog-> azidothymidine (AZT) – thymine analog

4444



The Acquisition of Drug Resistance

Adaptive responseAdaptive response in which microorganisms begin to in which microorganisms begin to toleratetolerate an amount of drug that would ordinarily be an amount of drug that would ordinarily be inhibitory; due to genetic versatility or variation; intrinsic inhibitory; due to genetic versatility or variation; intrinsic and acquiredand acquired

Acquired resistanceAcquired resistance::• spontaneous mutationsspontaneous mutations in critical chromosomal genes in critical chromosomal genes• acquisition of new genesacquisition of new genes or sets of genes or sets of genes via transfervia transfer

from another speciesfrom another species originates from originates from resistance factors (plasmids)resistance factors (plasmids) encoded with encoded with

drug resistance, transposonsdrug resistance, transposons

4545




Resistance factor (plasmid coded) transfered via conjugation or transfection

4646

Mechanisms of Drug Resistance

4747




Natural Selection and Drug ResistanceNatural Selection and Drug Resistance

Large populations of microbesLarge populations of microbes likely to likely to include drug resistant cellsinclude drug resistant cells due to due to prior mutations or transfer of plasmids – no growth advantage until exposed prior mutations or transfer of plasmids – no growth advantage until exposed to drugto drug

If exposedIf exposed, sensitive cells are inhibited or destroyed while , sensitive cells are inhibited or destroyed while resistance cells resistance cells will survivewill survive and proliferate. and proliferate.

Eventually population will be resistant – selective pressure - Eventually population will be resistant – selective pressure - natural natural selectionselection..

Worldwide indiscriminate use of antimicrobials has led to Worldwide indiscriminate use of antimicrobials has led to explosion of drug explosion of drug resistant microorganismsresistant microorganisms..

4848


Interaction between Drug and HostInteraction between Drug and Host

Estimate that 5% of all persons Estimate that 5% of all persons taking antimicrobials will taking antimicrobials will experience a serious adverse experience a serious adverse reaction to the drug – reaction to the drug – side side effectseffects

Major side effects:Major side effects:• direct damage to tissue due direct damage to tissue due

to to toxicity of drugtoxicity of drug • allergic reactionsallergic reactions• disruption in the balance of disruption in the balance of

normal flora- normal flora- superinfectionssuperinfections possiblepossible

Superinfections

5151


Considerations in Selecting an Antimicrobial DrugConsiderations in Selecting an Antimicrobial Drug

Identify the microorganismIdentify the microorganism causing the infection. causing the infection. -> test as soon as possible-> test as soon as possible

-> sample taken before antimicrobials are initiated-> sample taken before antimicrobials are initiated

Test the microorganism’s Test the microorganism’s susceptibility (sensitivity) to susceptibility (sensitivity) to various drugsvarious drugs in vitroin vitro when indicated. when indicated.

-> -> Kirby-Bauer disk diffusion testKirby-Bauer disk diffusion test

-> Dilution tests – -> Dilution tests – minimum inhibitory concentration (MIC)minimum inhibitory concentration (MIC) - - smallest concentration of drug that visibly inhibits growthsmallest concentration of drug that visibly inhibits growth

5252


Considerations in Selecting an Antimicrobial DrugConsiderations in Selecting an Antimicrobial Drug

Testing for Drug Susceptibility - The MIC and Therapeutic IndexTesting for Drug Susceptibility - The MIC and Therapeutic Index

In vitroIn vitro activity of a drug is not always correlated with activity of a drug is not always correlated with in vivo in vivo effect.effect.• If therapy fails, a different drug, combination of drugs, or If therapy fails, a different drug, combination of drugs, or

different administration must be considered.different administration must be considered.

Best to choose a drug with Best to choose a drug with highest level of selectivityhighest level of selectivity but but lowest level toxicitylowest level toxicity – measured by – measured by therapeutic indextherapeutic index – – the the ratio of the dose of the drug that is toxic to humans as ratio of the dose of the drug that is toxic to humans as compared to its minimum effective dosecompared to its minimum effective dose

High indexHigh index is desirable. is desirable.

1 applied microbiology 9 th lecture: chapter 12. 2 the elements of chemotherapy principles of...

Documents

streptomyces slide

microbe antimicrobial

vertebrate host cell

drug narrowspectrum

bacterial cell walls

specific cell component

discovery of penicillin

toxic drugs