Testing Antibiotics & Disinfectants

Kirby Bauer Disk Diffusion Technique

Antibiotics & Disinfectants   Perform lawn inoculation of
bacterium
  Apply disk containing antimicrobial
chemical and incubate
  Measure zone of inhibition
  A zone of inhibition indicates
antibiotic is effective and bacterium
is susceptible to the antibiotic
  If there is no zone of inhibition, the
antibiotic is not effective and the
bacterium is resistant to the antibiotic

Main Categories of Antibiotics Cell Wall Inhibitors

  Cell wall inhibitors Cell wall inhibitors target peptidoglycan
  Target peptidoglycan
.
  Protein synthesis inhibitors
  Target 70S ribosomes
  Nucleic acid synthesis inhibitors
  Prevent bacterial DNA or RNA synthesis
  Structural analogs
  Inhibit synthesis of essential metabolites

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Cell Wall Inhibitor Cell Wall Inhibitors
  Penicillins   Penicillin
  The active site on the
  Cephalosporins penicillin molecule is
  Carbepenems the β lactam ring
  The β lactam ring
  Polypeptide antibiotics prevents peptide cross
linkage in
peptidoglycan
  Some bacteria
produce β lactamase
and are resistant to
β lactam antibiotics

Cell Wall Inhibitors Cell Wall Inhibitors

  Newer Penicillins   Cephalosporins
  Ampicillin® and Amoxicillin® are examples   Cephalonthin (Ceclor® ) is an example
  One reason they are broader spectrum is   The active site is the β lactam ring
because they can get through the outer
membrane of gram negative bacteria   Stable to β lactamase
  Also, the β lactam ring is protected by other
atoms in the molecule
  Stable to β lactamase

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Cell Wall Inhibitors Cell Wall Inhibitors
  Carbapenems   Polypeptide antibiotics - effective against G+ bacteria
  Bacitracin®
  Imipenem (Primaxin®) is an example   Interferes with NAG-NAM bonds of peptidoglycan
  The active site is the β lactam ring   Ototoxic and nephrotoxic
  Topical use only
  Stable to β lactamase
  Vancomycin®
  Used for serious infections   Interferes with NAG-NAM bonds of peptidoglycan
  Usually administered intravenously   Ototoxic and nephrotoxic
  Used for gram positive bacteria that are resistant to other
  Can cause seizures antibiotics
  Usually administered intravenously

Cell Wall Inhibitors

Protein Synthesis Inhibitors
  Penicillin family - β lactam ring
target peptidoglycan cross linkage
  Bind to 70S
  Penicillin – vulnerable to β lactamase ribosomes and
  Amoxicillin - semisynthetic, stable to β lactamase prevent protein
  Cephalosporins - β lactam ring, stable to β lactamase
target peptidoglycan cross linkage synthesis. Do not bind
  Cephalothin (Ceclor) to 80S ribosomes in
  Carbepenems - β lactam ring, stable to β lactamase
target peptidoglycan cross linkage
eukaryotic cells
  Imipenem (Primaxin) - has potential side effects
  Polypeptide antibiotics
target NAG-NAM bonds
  Bacitracin – topical use only
  Vancomycin - has potential side effects

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Protein Synthesis Inhibitors Protein Synthesis Inhibitors
  Aminoglycosides   Aminoglycosides
  Macrolides   Streptomycin and gentamycin are examples
  Broad spectrum
  Tetracyclines   Gentamycin is effective against hard to kill
  Oxazolidonones G- bacteria like Pseudomonas
  Bind to 70S ribosomes
  Chloramphenicol
  Ototoxic (hearing loss) and nephrotoxic (kidney
  Streptogramins damage)
  Usually administered I.V. or I.M.

Protein Synthesis Inhibitors Protein Synthesis Inhibitors

  Macrolides   Tetracyclines
  Erythromycin and azithromycin (Z-pack) are   Doxycycline and Minocycline are examples
examples   Broad spectrum
  Broad spectrum   Bind to 70S ribosomes
  Bind to 70S   Binds to calcium and permanently discolors
  Few side effects developing teeth and bones.
  Not used in pregnant women and young
children

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Protein Synthesis Inhibitors Protein Synthesis Inhibitors
  Oxazolidinones   Chloramphenicol
  Zyvox is an example   Broad spectrum
  Broad spectrum   Bind to 70S ribosomes
  Bind to 70S ribosomes   Can cause aplastic anemia
  Used for serious infections such as MRSA

Protein Synthesis Inhibitors Nucleic Acid Replication Inhibitors

  Streptogramins   Quinolones
  Synercid   Cipro is an example
  Used for serious life-threatening infections   Broad spectrum
caused by vancomycin resistant bacteria   Interferes with DNA gyrase
  Bind to 70S ribosomes   Possible side effects: tendonitis leading to
  Administered by I.V. tendon rupture

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Nucleic Acid Replication Inhibitors
  Rifamycin
  Rifampin is an example
  Broad spectrum
  Inhibits mRNA synthesis
  Good tissue penetration and can cross the
blood/brain barrier
  Causes orange secretions
Antibiotic Resistance Mechanisms
  Destruction or inactivation of the drug
  Example: some bacteria produce β lactamase that
inactivates the β lactam ring
  Target modification
  Example: some bacteria produce modified ribosomes
that prevent macrolide attachment
  Rapid ejection of the drug
  Example: some gram negative bacteria eject antibiotics
that get past the outer membrane
Structural Analogs
  Septra is an example
  Sulfonamide/Trimethoprim combination
  Broad spectrum
  Inhibits dihydrofolate reductase
  Used in urinary tract and ear infections
Antibiotic Resistance
  Resistant strains emerge ……
  through selection of resistant strains in the environment
  or the exchange of genetic material between bacteria
  Transfer of genetic material in gram positive
bacteria such as Staphylococcus usually occurs
through transduction
  Transfer of genetic material in gram negative
bacteria such as E.coli usually occurs through
conjugation
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Antibiotic Resistance Antibiotic Resistance
  The first day of   Day 3 of antibiotic
antibiotic therapy therapy
  Some bacteria will be   Most of the susceptible
highly susceptible, bacteria and many of
some will be highly the intermediate
resistant and most will bacteria have been
have an intermediate killed. The resistant
response bacteria are not
affected.

Antibiotic Resistance Antibiotic Resistance

  Day 6 of antibiotic   Day 10 of antibiotic
therapy therapy
  More of the   All bacteria are killed
intermediate bacteria
have been killed. The
resistant bacteria are
minimally affected.

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Antibiotic Resistance Main Categories of Disinfectants
  Here is what happens if   Phenols - damage cell membrane
the patient discontinues   Cresols (Lysol)
antibiotic therapy on   Hexachlorophene (pHisoHex)
day 6   Trichlosan (in many antiseptics)
  Halogens – denature proteins
  Iodine (Betadine)
  Chlorine (Chlorox)
  Alcohols – denature proteins and disrupt membranes
  Isopropyl – 70% (rubbing alcohol)
  Ethanol – 70% (Purell)

Main Categories of Disinfectants Main Categories of Disinfectants

  Heavy metals – denature proteins   Oxidizing agents – denature proteins
  Silver (Silvadene cream)   H2O2
  Mercury (thimersol)   Benzoyl peroxide
  Zinc (Desitin)   Ozone
  Quaternary ammonium compounds – cell membrane   Ethylene oxide gas – denatures proteins
Surface active cationic detergents (+ charge)
  Very effective
  Benzalkonium chloride
  Sterilizes
  Cetylpyridinium chloride (Cepacol)
  Aldehydes – denature proteins
  Formaldehyde