Microbial Growth Control

1
Chapter 20

• Microbial Growth Control

2
Physical Antimicrobial Control

• 3 types
• (1) heat sterilization
• (2) radiation sterilization
• (3) filter sterilization

3
Heat Sterilization

• Denaturation of cellular macromolecules ? death
• Death exponential
• Sterilization requires lower temps. for a longer
  period of time.
• Moist heat is better than dry heat for
  sterilization why?
• Thermal death time time is takes to kill all
  cells at a given time.

4
Endospores

• Heat sterilization procedures are designed to
  destroy endospores.
• A major factor in the resistance of endospores is
  the amount and state of water they contain
  (minimal amounts and becomes gel-like).
• Only 0.5 min. and 65C required at 15 psi to kill
  vegetative cells.

5
The Autoclave/Pasteurization

• The Autoclave
• Uses high temp. and high pressure.
• 121C, 15 psi, 15 min.
• Pasteurization
• Reduces microbes in milk and other heat-sensitive
  foods.
• ? sterilization b/c not all org. are killed.
• Prevents spread of pathogens and food spoilage.
• 71C, 15 sec.

6
Radiation Sterilization

• Nonionizing radiation (UV light) is germicidal
  and used to sterilize exposed surfaces, but
  cannot penetrate deeper.
• Ionizing radiation (ex. x-rays) produces reactive
  molecules, which degrade DNA. Ionizing radiation
  also directly causes breaks in DNA. Used for
  sterilization and decontamination in the medical
  supplies and food industries.

7
Filter Sterilization

• (1) Depth filter fibrous mat that traps
  particles in paths created throughout the depth
  of the filter often used as prefilters.
• (2) Membrane filter functions more like a
  sieve, trapping many particles on the filter
  surface, size of the holes and molecules passing
  through are precisely controlled, used to
  sterilize liquids.
• (3) Nucleopore filter sizes of the holes is
  precisely controlled, used in EM to remove and
  concentrate a sample from a liquid.

8
Chemical Growth Control

• Antimicrobial agents natural or synthetic
  chemical that kills or inhibits the growth of
  microorganisms.
• Cidal agents that kill organisms without
  causing lysis, ex. fungicidal.
• Static agents that inhibit the growth of
  organisms, ex. viristatic.
• Lytic agents that cause cell lysis, ex.
  bacteriolytic.
• Minimum Inhibitory Concentration (MIC) smallest
  amount of an agent needed to inhibit the growth
  of an organism, determined by the broth dilution
  method.

9
Antiseptics, Disinfectants, and Sterilants

• Antiseptics chemical agents that kill or
  inhibit growth of microorganisms and are nontoxic
  enough to be applied to living tissues.
• Disinfectants (germicides) chemicals that kill
  microorganisms and are used on inanimate objects.
• Sterilants disinfectants that can kill all
  microbial life and are used to sterilize
  inanimate objects and surfaces.

10
Synthetic Antimicrobial Drugs

• These chemotherapeutic agents have selective
  toxicity ability to inhibit bacteria or other
  pathogens without harming the host.
• Growth factor analogs Analogs block the
  utilization of growth factors required by
  pathogens, analogs of vitamins, amino acids, etc.
  Ex. of an analog sulfa drugs, which block
  synthesis of a nucleic acid precursor.
• Quinolones interact with DNA gyrase, preventing
  it from supercoiling DNA.

11
Naturally Occurring Antimicrobial Drugs
Antibiotics

• Antibiotics natural chemical compounds produced
  by microorganisms that inhibit or kill other
  microorganisms.
• Less than 1 of antibiotics are of practical
  value in medicine.
• Broad spectrum antibiotic works on gram-pos.
  and gram-neg. bacteria.
• Targets of antibiotics cell wall (vancomycin),
  cytoplasmic membrane (polymyxins), protein
  synthesis (macrolides and tetracyclines), and
  nucleic acid synthesis (rifampin).
• Antibiotics inhibiting protein synthesis by
  interacting with the ribosome.

12
?-Lactam Antibiotics Penicillins and
Cephalosporins

• ?-Lactam Antibiotics penicillins, etc. All
  share a ?-lactam ring.
• Natural penicillin penicillin G, active against
  gram-positive bacteria.
• Semisynthetic penicillin is also effective
  against gram-neg. bacteria.
• Cephalosporins also have a ?-lactam ring, but
  are structurally different from the penicillins.
  

13
Mechanisms of Action

• ?-lactam antibiotics inhibit cell wall synthesis
  by interfering with the transpeptidation reaction
  of peptidoglycan synthesis.
• These antibiotics act as PG precursor analogs
  that bind to the transpeptidase enzyme.
• Transpeptidases are also called penicillin
  binding proteins (PBPs) since they bind to
  penicillin, when it is present, instead of
  binding to PG precursors and forming the cell
  wall.
• During PG synthesis autolysins have been
  activated to make cuts to expand the cell wall,
  but penicillin inhibits the process of adding in
  new pieces. The autolysins continue to work,
  even though the transpeptidase has been inhibited
  by penicillin.

14
Antibiotics from Prokaryotes

• Aminoglycoside antibiotics ex. streptomycin,
  inhibit protein synthesis at the ribosome, used
  against gram-neg. bacteria, has serious side
  effects, bacterial resistance readily develops,
  used as a last resort when other antibiotics
  fail.
• Macrolide antibiotics ex. erythromycin,
  inhibits protein synthesis at the ribosome, used
  clinically in place of penicillin in patients
  allergic to penicillin.
• Tetracyclines broad-spectrum antibiotics
  (inhibit almost all gram-pos. and gram-neg.
  bacteria), protein synthesis inhibitor, widely
  used in human and veterinary medicine, widespread
  resistance is a concern.

15
Antiviral Drugs

• Antiviral Chemotherapeutic Agents the most
  successful and commonly used are nucleoside
  analogs, ex. AZT blocks synthesis of the
  DNAintermediate (reverse transcription). These
  drugs exhibit some level of host toxicity.
  Protease inhibitors inhibit HIV infection by
  binding to the active site of the HIV protease.
• Interferons antiviral substances produced by
  animal immune cells (lymphocytes) that promote
  the synthesis of antiviral proteins that prevent
  further virus infection. Interferons are not
  virus-specific, but are host-specific.

16
Antifungal Drugs

• Fungi are euk. and, thus, present a special
  problem for treatment.
• Ergosterol inhibitors interfere with ergosterol
  or inhibit its synthesis. Ergosterol replaces
  cholesterol found in animal cell membranes,
  causing the fungal cell membrane to remain fluid.
  These inhibitors result in membrane damage
  structually and functionally.
• Other antifungal agents inhibit chitin (cell wall
  component) biosynthesis, folate biosynthesis, DNA
  replication, ir mitosis.
• There is concern about the emergence of
  antifungal resistant fungi, ex. Candida.

17
Antimicrobial Drug Resistance

• Antimicrobial drug resistance acquired ability
  of an organism to resist the effects of a
  chemotherapeutic agent to which it is normally
  susceptible.
• Most antimicrobial resistance involves resistance
  genes that are transferred by means of genetic
  exchange. Antibiotic producers develop
  resistance mechanisms to enable them to be
  resistant to their own antibiotics.

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Resistance Mechanisms

• Reasons for resistance
• (1) organism lacks the structure an antibiotic
  inhibits what is an example of this?
• (2) organism may be impermeable to the antibiotic
  what is an example of this?
• (3) organism may be able to alter the antibiotic
  to an inactive form what is an example of this?
• (4) organism may modify the target of the
  antibiotic
• (5) by genetic change, alteration may occur in a
  metabolic pathway that the antimicrobial agent
  blocks, so that the organism develops a resistant
  biochemical pathway.
• (6) organism may be able to pump out the
  antibiotic entering the cell (efflux).

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Resistance Mediated by R Plasmids vs. Chromosomes

• Antibiotic resistance may be chromosomal or
  plasmid-encoded.
• Antibiotic resistance mediated by chromosomal
  genes arises because of a modification of the
  target of antibiotic action.
• R plasmid resistance is in most cases due to the
  presence in the R plasmid of genes encoding new
  enzymes that inactivate the drug or genes that
  encode enzymes that either prevent uptake of the
  drug or actively pump it out.
• R plasmids existed in natural microbial
  populations before antibiotics, and even
  synthetic antibiotics, were discovered and used.

20
Spread of Antimicrobial Drug Resistance

• Inappropriate, extensive use of antimicrobial
  drugs is leading to the rapid development of
  drug-resistance in disease-causing
  microorganisms.
• Drugs prescribed for treatment of a particular
  infection have changed because of increased
  resistance of the microorganism causing the
  disease.
• Ex. Neisseria gonorrhoeae is now resistant to
  penicillin.
• Antibiotic treatment is warranted in 20 of
  individuals who are seen for clinical infectious
  disease, yet antibiotics are prescribed up to 80
  of the time. In up to 50 of cases recommended
  doses or duration of treatments are not correct.
• This is compounded by patient noncompliance how?

21
Spread of Antimicrobial Drug Resistance (cont.)

• Other indiscriminant, nonessential uses of
  antibiotics contribute to the emergence of
  resistant strains, ex. antibiotics are used in
  agriculture both a growth-promoting substances in
  animal feeds and as prophylactics what does
  that mean?
• If the use of a particular antibiotic is stopped,
  resistance to that antibiotic may be reversed
  over time.

22
The Search for New Antimicrobial Drugs

• The production of new analogs of existing
  antimicrobial compounds is often productive since
  they mimic the original drugs to some extent and
  have a predictable mechanism of action, but may
  be different enough to act on organisms resistant
  to the original form of the drug.
• Application of automated robotic chemistry
  methods to drug discovery combinatorial
  chemistry.
• Many different derivatives of an antimicrobial
  agent can be generated in a short time, ex. 725
  different tetracycline derivatives from only 6
  different reagents in only a few hours.
• According to the pharmaceutical industry, 7
  million candidate compounds must be screened to
  yield a single useful clinical drug. New drug
  discovery 10-25 years and 500 million for each
  new drug approved (FDA).
• Computerized drug design can facilitate the
  design of completely new drugs, ex. a protease
  inhibitor currently used to treat HIV.