MCB 3020, Spring 2005
Microbial Growth Control
and Antibiotic Resistance
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Chapter 18 Microbial Growth Control:
I. Microbial growth control
II. Measuring antimicrobial activity
III. Food preservation
IV. Antimicrobial drugs
V. Antibiotic Resistance
I. Microbial Growth Control
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A. uses
B. autoclave
C. radiation
D. filters
E. chemical agents
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I. Microbial Growth Control
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Sterilization
Killing or removal of all living
organisms and their viruses
Inhibition
limiting microbial growth
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A. Uses
Food preservation
Laboratory work
Disease prevention
Disease treatment
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B. Autoclave
Machine that uses
steam under pressure
for sterilization.
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Autoclave 8
Items are heated to
121°C for 10-15 minutes.
kills endospores
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C. Radiation
1. Ultraviolet (220 to 300 nm)
used to disinfect surfaces and air
poor penetrating power
2. Gamma and X-rays
ionizing radiation
used for food preservation and
sterilization of surgical supplies
good penetration
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D. Filters
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Used to sterilize heat-sensitive
solutions and gasses
A pore size of 0.22 micron will
remove most bacteria.
Will it remove most viruses?
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E. Chemical Agents
Antimicrobial agents
Chemicals that kill or inhibit the
growth of microorganisms
Cidal agents
Chemicals that kill
(bacteriocidal,
fungicidal, viricidal)
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Bacteriostatic agents
chemicals that inhibit growth,
but do not kill
frequently are inhibitors of
protein synthesis
Bacteriolytic agents
kill cells by lysis
eg. penicillin
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1. Disinfectants
Chemicals used to kill microbes on
inanimate objects.
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Chlorine
Phenolic compounds
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2. Antiseptics
Chemicals used to kill microbes on
living tissue.
Alcohol (70% on skin)
Hydrogen peroxide
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II. Measuring antimicrobial activity
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A.Tube dilution assay
B. Agar diffusion assay
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A. Tube dilution assay
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1. inoculate tubes containing several
concentrations of test compound with
test organism and incubate.
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bacterial
growth
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.1
.01
.001
MIC
MIC (minimum inhibitory concentration)
The lowest concentration of a substance
that inhibits growth of a test organism TB
B. Agar diffusion assay
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lawn of test bacteria
filter paper soaked
with test compound
agar plate
zones of inhibition
(no growth)
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III. Food preservation
A. Common spoilage organisms
B. Preservation methods
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A. Common food spoilage organisms
Meat
enteric bacteria
Escherichia coli
Salmonella
Milk products
lactic acid bacteria
Fruits and vegetables
Erwinia
Pseudomonas
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B. Preservation methods
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1. Pasteurization
Heat treatment to reduce the
number of viable organisms.
71°C, 15 sec., or 63-66°C, 30 min
Used on foods the would be ruined
by higher temperatures.
milk
juice
wine
etc.
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2. Temperature control
refrigeration and freezing
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3. Sterilization
canning
4. Controlling water availability
adding salt (eg. ham)
adding sugar (eg. jelly)
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5. pH control
pickling
fermented foods
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6. Chemical preservatives
Na propionate
Na benzoate
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IV. Antimicrobial drugs
A. Selective toxicity
B. Growth factor analogs
C. Antibiotics
D. Antivirals and antifungals
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A. Selective toxicity
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Toxicity for the pathogen,
but not for the host.
Something to think about:
What is the basis for selective
toxicity?
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B. Growth factor analogs
A substance structurally
related to a growth factor
that blocks its use.
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1. Sulfanilamide
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Growth factor analog structurally
related to p-aminobenzoic acid
(PABA)
Inhibits microbial growth by
inhibiting folate synthesis
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H2N
SO2NH2
H2N
PABA
sulfanilamide
HN
H2N
COOH
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N
N N
CH2
H
N
O
C
R
folate
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Sulfanilamide is nontoxic
to humans because we take up
folate from our diet.
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C. Antibiotics
Substances produced by microbes
that kill or inhibit the growth of
microbes
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bacteriocidal agents kill
bacteriostatic agents inhibit growth
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1. inhibitors of cell wall synthesis
penicillin
vancomycin
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2. inhibitors protein synthesis
erythromycin (50S ribosomal subunit)
tetracycline (30S ribosomal subunit)
streptomycin (30S ribosomal subunit)
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R
H
N
S
CH3
H
O
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CH3
N
COOH
beta-lactam ring
natural penicillin R =
CH2-CO-
Prevents transpeptidation in cell wall
Erythromycin (macrolide)
CH3
H3C
O
HO
H3C
H3C
H2C
H2C
CH3
OH
HO
HO
O
O
CH3
O
O
macrolide ring
N
O
CH3 O
H3C
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CH3
CH3
CH3
OH
OCH3
50S ribosomal subunit
R4
OH
H3C
R2 R3 R1 H
O
OH
OH
N
CH3
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4
OH
CO
O
NH2
Tetracycline: R1=H, R2=OH, R3=CH3, R4=H
• Broad spectrum
• Target: 30s ribosomal subunit
Kanamycin (aminoglycoside)
H2C-NH2
O
HO
OH
NH2
O NH2
OH
O
OH
CH2OH
O
HO
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NH2
OH
O
Target:
30 s ribosomal
subunit
3. inhibitors of DNA gyrase
naladixic acid
novobiocin
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4. inhibitors of RNA synthesis
rifampin
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Antivirals and Antifungals
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A. Antivirals
Chemicals
rifampin
azidothymidine (AZT)
Interferon
inhibits viral RNA synthesis
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B. Antifungals
Ergosterol inhibitors
polyenes
azoles
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Important point
Selective toxicity is more difficult to
obtain with antivirals and antifungals
Why?
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V. Antibiotic Resistance
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A. the problem of resistance
B. resistance mechanisms
C. development of resistance
D. enzymes that inactivate antibiotics
A. The problem of resistance
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Examples of drug-resistant bacteria
• Vancomycin-resistant Staphlyococcus
aureus
• Penicillin-resistant Streptococcus
pneumoniae
• Quinolone-resistant Salmonella enterica
Why so much resistance?
• Overuse of antibiotics in inpatient and
outpatient settings.
• Increased use of quinolones, tetracyclines,
and glycopeptides in agriculture, the poultry
industry, veterinary practice, and marine
biology.
• Newer, implantable cardiovascular and
orthopedic devices that necessitate
prophylactic antibiotics.
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B. Resistance mechanisms
• lack of target site
• impermeability
• chemical modification
of the antibiotic
• pump antibiotic out of cell
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C. Development of resistance
1. Mutation
target site modification
2. Gene transfer
R-plasmids
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R-plasmids (resistance plasmids)
Plasmids that carry antibiotic
resistance genes.
Antibiotic resistance genes usually
encode enzymes that
inactivate antibiotics
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D. Enzymes that inactivate antibiotics
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1. Chloramphenicol acetyltransferase
acetylates chloramphenicol
2. beta-lactamase
cleaves the beta-lactam ring
3. Tetracycline pump
pumps tetracycline out of the cell
Interactions of antibiotics with alcohol
in humans
• Antibiotics that are affected by alcohol are
chloramphenicol, cephalosporins,
metronidazole, and others.
• These produce "disulfiram-like" reactions.
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Disulfiram-like reactions
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• Disulfiram is a drug to treat alcoholism.
• Some antibiotics cause a reaction similar to disulfiram
reactions.
• Inactivates the enzyme aldehyde dehydrogenase.
• Causing accumulation of acetaldehyde in blood.
• Symptoms are flushed face, severe headaches, chest
pains, shortness of breath, vomiting, and sweating.
Ethanol
Alcohol
dehydrogenase
NAD+
NADH
Acetaldehyde
NAD+
NADH
X
Aldehyde
dehydrogenase
Acetate
Acetyl CoA
Disulfiram
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Study objectives
1. Know how the following are used to control microbial growth: autoclaves, 97
radiation, filters, disinfectants, antiseptics. Contrast cidal agents,
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bacteriostatic agents,and lytic agents. Know the examples presented in class.
2. Compare and contrast the tube dilution assay and the agar diffusion assay.
Understand how each is used to measure antimicrobial activity of chemicals.
3. What is MIC? How does it correlate with antimicrobial activity of an inhibitor?
4. Memorize the common food spoilage organisms covered in class.
5. Know what pasteurization is and what types of foods are pasteurized.
6. Memorize the food preservation methods and examples presented in class.
7. What is selective toxicity? Understand the basis of selective toxicity of growth
factor analogs, antibiotics, azidothymidine, interferon, and antiviral agents.
8. What is a growth factor analog?
9. How does sulfanilamide inhibit the growth of some bacteria?
10. Know the names and targets of the antibiotics presented in lecture.
11. Know the following antiviral agents:rifampin, azidothymidine, interferon.
Azidothymidine inhibits reverse transcriptase. What viruses are affected this?
12. Know the names and target of the antifungal agents (polyenes, azoles).
13. Why is selective toxicity more difficult to obtain with antivirals and antifungals?
Study objectives
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14. Give 3 examples of antibiotic-resistant bacteria.
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What are the major causes of antibiotic resistance?
15. What are the mechanisms by which bacteria require antibiotic resistance?
What is the role of R-plasmids in resistance?
Understand how antibiotic-inactivating enzymes work.
16. Explain why alcohol should not be consumed while taking some antibiotics.
Why do disulfiram-like antibiotics cause symptoms when consumed with
alcohol?