Applied Microbiology

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Applied Microbiology

• 9th lecture Chapter 12

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The Elements of Chemotherapy
Principles of Antimicrobial Therapy
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Origins of Antibiotic Drugs
The Elements of Chemotherapy
Principles of Antimicrobial Therapy
Alexander Fleming 1928 discovery of Penicillin
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Origins of Antibiotic Drugs
The Elements of Chemotherapy
Principles of Antimicrobial Therapy

• Antibiotics are common metabolic products of
  aerobic spore-forming bacteria and fungi.
• bacteria in genera Streptomyces and Bacillus
• molds in genera Penicillium and Cephalosporium
• By inhibiting the other microbes in the same
  habitat, antibiotic producers have less
  competition for nutrients and space.

Streptomyces
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The Elements of Chemotherapy
Principles of Antimicrobial Therapy
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The Elements of Chemotherapy
Interactions between Drug and Microbe

• Antimicrobial drugs should be selectively toxic -
  drugs should kill or inhibit microbial cells
  without simultaneously damaging host tissues.
• As the characteristics of the infectious agent
  become more similar to the vertebrate host cell,
  complete selective toxicity becomes more
  difficult to achieve and more side effects are
  seen.

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Mechanisms of Drug Action
The Elements of Chemotherapy
Interactions between Drug and Microbe
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Mechanisms of Drug Action
The Elements of Chemotherapy
Interactions between Drug and Microbe
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Mechanisms of Drug Action Spectrum of Drug
The Elements of Chemotherapy
Interactions between Drug and Microbe

• Spectrum range of activity of a drug
• narrow-spectrum effective on a small range of
  microbes
• target a specific cell component that is found
  only in certain microbes
• broad-spectrum greatest range of activity
• target cell components common to most pathogens

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Mechanisms of Drug Action Cell Wall Synthesis
The Elements of Chemotherapy
Interactions between Drug and Microbe

• Most bacterial cell walls contain peptidoglycan.
• Penicillins and cephalosporins block synthesis of
  peptidoglycan, causing the cell wall to lyse.
• Active on young, growing cells
• Penicillins do not penetrate the outer membrane
  and are less effective against Gram-negative
  bacteria.
• Broad spectrum penicillins and cephalosporins can
  cross the cell walls of Gram-negative bacteria.

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Mechanisms of Drug Action Cell Wall Synthesis
The Elements of Chemotherapy
Interactions between Drug and Microbe
Healthy cell
Drug affected cell
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Mechanisms of Drug Action Cell Wall Synthesis
The Elements of Chemotherapy
Interactions between Drug and Microbe
Penicillin and Cephalosporin block peptidase to
link NAMs
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Mechanisms of Drug Action Disruption of
Membrane Function
The Elements of Chemotherapy
Interactions between Drug and Microbe

• A cell with a damaged membrane dies from
  disruption in metabolism or lysis.
• These drugs have specificity for a particular
  microbial group, based on differences in types of
  lipids in their cell membranes.
• Polymyxins interact with phospholipids and cause
  leakage, particularly in Gram-negative bacteria.
• Amphotericin B and nystatin form complexes with
  sterols on fungal membranes which causes leakage.
• -gt Antimicrobial Peptides !!!

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Mechanisms of Drug Action Disruption of
Membrane Function
The Elements of Chemotherapy
Interactions between Drug and Microbe
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Mechanisms of Drug Action Inhibition of DNA and
RNA Synthesis
The Elements of Chemotherapy
Interactions between Drug and Microbe

• May block synthesis of nucleotides, inhibit
  replication, or stop transcription
• Chloroquine binds and cross-links the double
  helix quinolones inhibit DNA helicases
  (Replication).
• Antiviral drugs that are analogs of purines and
  pyrimidines insert in viral nucleic acid,
  preventing replication.

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Mechanisms of Drug Action Inhibition of DNA and
RNA Synthesis
The Elements of Chemotherapy
Interactions between Drug and Microbe
DNA Synthesis Cell devision-gt DNA Replication
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Mechanisms of Drug Action Inhibition of DNA and
RNA Synthesis
The Elements of Chemotherapy
Interactions between Drug and Microbe
DNA Replication One strand template for new
strand -gt Replication
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Mechanisms of Drug Action Inhibition of DNA and
RNA Synthesis
The Elements of Chemotherapy
Interactions between Drug and Microbe
RNA Synthesis -gt Transcription mRNA molecule
made from DNA
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Mechanisms of Drug Action Inhibition of Protein
Synthesis
The Elements of Chemotherapy
Interactions between Drug and Microbe

• Ribosomes of eukaryotes differ in size and
  structure from prokaryotes antimicrobics usually
  have a selective action against prokaryotes can
  also damage the eukaryotic mitochondria
• Aminoglycosides (streptomycin, gentamycin) insert
  on sites on the 30S subunit and cause misreading
  of mRNA.
• Tetracyclines block attachment of tRNA on the A
  acceptor site and stop further synthesis.

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Mechanisms of Drug Action Inhibition of Protein
Synthesis
The Elements of Chemotherapy
Interactions between Drug and Microbe
Protein Synthesis -gt Translation mRNA molecule
used to make protein
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Mechanisms of Drug Action Inhibition of Pathways
The Elements of Chemotherapy
Interactions between Drug and Microbe

• Sulfonamides and trimethoprim block enzymes
  required for tetrahydrofolate synthesis needed
  for DNA and RNA synthesis.
• Competitive inhibition drug competes with
  normal substrate for enzymes active site
• Synergistic effect an additive effect, achieved
  by multiple drugs working together, requiring a
  lower dose of each

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Mechanisms of Drug Action Inhibition of Pathways
Interactions between Drug and Microbe
Competitive Inhibition of Sulfonamides
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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups

• Antibacterial drugs
• antibiotics
• synthetic drugs
• Antifungal drugs
• Antiprotozoan drugs
• Antiviral drugs
• -gt About 260 different antimicrobial drugs are
  classified in 20 drug families.

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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antibacterial drugs acting on cell wall gt
ß-lactam antibiotics

• Large diverse group of compounds
• Could be synthesized in the laboratory
• More economical to obtain natural penicillin
  through microbial fermentation and modify it to
  semi-synthetic forms
• Penicillium chrysogenum major source
• All consist of 3 parts
• thiazolidine ring
• beta-lactam ring
• variable side chain dictating microbial activity

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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antibacterial drugs acting on cell wall gt
ß-lactam antibiotics

• Beta-lactam antimicrobials - all contain a highly
  reactive 3 carbon, 1 nitrogen ring -gt ß-lactam
  ring
• Primary mode of action is to interfere with cell
  wall synthesis.
• Greater than ½ of all antimicrobic drugs are
  beta-lactams.
• Penicillins and cephalosporins most prominent
  beta-lactams.

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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antibacterial drugs acting on cell wall gt
ß-lactam antibiotics (Penicillins)
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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antibacterial drugs acting on cell wall gt
ß-lactam antibiotics (Penicillins)
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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antibacterial drugs acting on cell wall gt
ß-lactam antibiotics (Penicillins)

• Penicillins G and V most important natural forms
• Penicillin is the drug of choice for
  Gram-positive cocci (streptococci) and some
  Gram-negative bacteria (meningococci and syphilis
  spirochete).
• Semisynthetic penicillins ampicillin,
  carbenicillin and amoxicillin have broader
  spectra Gram-negative enteric rods
• Penicillinase-resistant methicillin, nafcillin,
  cloxacillin
• Primary problems allergies and resistant
  strains of bacteria

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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antibacterial drugs acting on cell wall gt
ß-lactam antibiotics (Cephalorins)

• Account for majority of all antibiotics
  administered
• Isolated from Cephalosporium acremonium mold
• Synthetically altered beta-lactam structure
• Relatively broad-spectrum, resistant to most
  penicillinases, cause fewer allergic reactions
• Some are given orally many must be administered
  parenterally.
• Generic names have root cef, ceph, or kef.

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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antibacterial drugs acting on cell wall gt
ß-lactam antibiotics (Cephalorins)

• 4 generations exist each group more effective
  against Gram-negatives than the one before with
  improved dosing schedule and fewer side effects
• first generation cephalothin, cefazolin most
  effective against Gram-positive cocci and few
  Gram-negative
• second generation cefaclor, cefonacid more
  effective against Gram-negative bacteria
• third generation cephalexin, ceftriaxone
  broad-spectrum activity against enteric bacteria
  with beta-lactamases
• fourth generation cefepime widest range both
  Gram- negative and Gram-positive

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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antibacterial drugs acting on cell wall gt
ß-lactam antibiotics (Cephalorins)
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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antibacterial drugs acting on cell wall gt non
ß-lactam antibiotics

• vancomycin narrow-spectrum, most effective in
  treatment of Staphylococcal infections in cases
  of penicillin and methicillin resistance or if
  patient is allergic to penicillin toxic and hard
  to administer restricted use
• bacitracin narrow-spectrum produced by a strain
  of Bacillus subtilis used topically in ointment
• isoniazid (INH) works by interfering with
  mycolic acid synthesis used to treat infections
  with Mycobacterium tuberculosis oral doses in
  combination with other antimicrobials such as
  rifampin, ethambutol

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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antibacterial drugs acting on Protein Synthesis -
Aminoglycosides

• Aminoglycosides composed of 2 or more amino
  sugars and an aminocyclitol (6C) ring binds
  ribosomal subunit
• Products of various species of soil actinomycetes
  in genera Streptomyces and Micromonospora
• Broad-spectrum, inhibit protein synthesis,
  especially useful against aerobic Gram-negative
  rods and certain gram-positive bacteria
• streptomycin bubonic plague, tularemia, TB
• gentamicin less toxic, used against
  Gram-negative rods
• newer tobramycin and amikacin Gram-negative
  bacteria

Streptomycin
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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antibacterial drugs acting on Protein Synthesis -
Tetracyclines

• Broad-spectrum, block protein synthesis by
  binding ribosomes
• Aureomycin, terramycin, tetracycline, doxycycline
  and minocycline low cost oral drugs side
  effects are a concern
• Treatment for STDs, Rocky Mountain spotted fever,
  Lyme disease, typhus, acne and protozoa

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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antibacterial drugs acting on Protein Synthesis -
Chloramphenicol

• Isolated from Streptomyces venezuelae no longer
  derived from natural source
• Potent broad-spectrum drug with unique
  nitrobenzene structure
• Blocks peptide bond formation
• Very toxic, restricted uses, can cause
  irreversible damage to bone marrow
• Typhoid fever, brain abscesses, rickettsial and
  chlamydial infections

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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antibacterial drugs acting on Protein Synthesis -
Macrolides

• Erythromycin large lactone ring with sugars
  attaches to ribosomal 50s subunit
• Broad-spectrum, fairly low toxicity
• Taken orally for Mycoplasma pneumonia,
  legionellosis, Chlamydia, pertussis, diphtheria
  and as a prophylactic prior to intestinal surgery
• For penicillin-resistant gonococci, syphilis,
  acne
• Newer semi-synthetic macrolides clarithomycin,
  azithromycin

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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antibacterial drugs blocking metabolic pathways

• Most are synthetic most important are
  sulfonamides, or sulfa drugs - first antimicrobic
  drugs
• Narrow-spectrum block the synthesis of folic
  acid by bacteria
• sulfisoxazole shigellosis, UTI, protozoan
  infections
• silver sulfadiazine burns, eye infections
• trimethoprim given in combination with
  sulfamethoxazole UTI, PCP

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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antifungal drugs

• Fungal cells are eukaryotic a drug that is toxic
  to fungal cells also toxic to human cells -gt more
  problematic
• Three examples out of five antifungal drug
  groups
• macrolide polyene - amphotericin B, nystatin
• synthetic azoles broad-spectrum ketoconazole,
  clotrimazole, miconazole
• flucytosine analog of cytosine cutaneous
  mycoses or in combination with amphotericin B for
  systemic mycoses

Polyenes
Azole
Flucytosine
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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antiviral drugs

• Selective toxicity is almost impossible due to
  obligate intracellular parasitic nature of
  viruses.
• Block penetration into host cell
• Block transcription or translation of viral
  genetic material
• nucleotide analogs
• acyclovir herpesviruses
• ribavirin- a guanine analog RSV, hemorrhagic
  fevers
• AZT thymine analog - HIV
• Prevent maturation of viral particles
• protease inhibitors HIV

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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
Antiviral drugs Drugs for treating HIV
infection and AIDS

• Retrovirus offers 2 targets for chemotherapy
• interference with viral DNA synthesis from viral
  RNA using nucleoside reverse transcriptase
  inhibitors (nucleotide analogs)
• interference with synthesis of DNA using
  nonnucleoside reverse transcriptase inhibitors
• -gt azidothymidine (AZT) thymine analog

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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
The Acquisition of Drug Resistance

• Adaptive response in which microorganisms begin
  to tolerate an amount of drug that would
  ordinarily be inhibitory due to genetic
  versatility or variation intrinsic and acquired
• Acquired resistance
• spontaneous mutations in critical chromosomal
  genes
• acquisition of new genes or sets of genes via
  transfer from another species
• originates from resistance factors (plasmids)
  encoded with drug resistance, transposons

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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
The Acquisition of Drug Resistance
Resistance factor (plasmid coded) transfered via
conjugation or transfection
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Mechanisms of Drug Resistance
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The Elements of Chemotherapy
Survey of Major Antimicrobial Drug Groups
The Acquisition of Drug Resistance
Natural Selection and Drug Resistance

• Large populations of microbes likely to include
  drug resistant cells due to prior mutations or
  transfer of plasmids no growth advantage until
  exposed to drug
• If exposed, sensitive cells are inhibited or
  destroyed while resistance cells will survive and
  proliferate.
• Eventually population will be resistant
  selective pressure - natural selection.
• Worldwide indiscriminate use of antimicrobials
  has led to explosion of drug resistant
  microorganisms.

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The Elements of Chemotherapy
Interaction between Drug and Host

• Estimate that 5 of all persons taking
  antimicrobials will experience a serious adverse
  reaction to the drug side effects
• Major side effects
• direct damage to tissue due to toxicity of drug
• allergic reactions
• disruption in the balance of normal flora-
  superinfections possible

Superinfections
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The Elements of Chemotherapy
Considerations in Selecting an Antimicrobial Drug

• Identify the microorganism causing the infection.
• -gt test as soon as possible
• -gt sample taken before antimicrobials are
  initiated
• Test the microorganisms susceptibility
  (sensitivity) to various drugs in vitro when
  indicated.
• -gt Kirby-Bauer disk diffusion test
• -gt Dilution tests minimum inhibitory
  concentration (MIC) - smallest concentration of
  drug that visibly inhibits growth

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The Elements of Chemotherapy
Considerations in Selecting an Antimicrobial Drug
Testing for Drug Susceptibility - The MIC and
Therapeutic Index

• In vitro activity of a drug is not always
  correlated with in vivo effect.
• If therapy fails, a different drug, combination
  of drugs, or different administration must be
  considered.
• Best to choose a drug with highest level of
  selectivity but lowest level toxicity measured
  by therapeutic index the ratio of the dose of
  the drug that is toxic to humans as compared to
  its minimum effective dose
• High index is desirable.