26.

1 Heat Sterilization
• Sterilization
– The killing or removal of all viable organisms
within a growth medium
• Inhibition
– Effectively limiting microbial growth
• Decontamination
– The treatment of an object to make it safe to
handle
• Disinfection
– Directly targets the removal of all pathogens, not
necessarily all microorganisms
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26.1 Heat Sterilization
• Heat sterilization is the most widely used method
of controlling microbial growth
• High temperatures denature macromolecules
– Amount of time required to reduce viability tenfold
is called the decimal reduction time
– Some bacteria produce resistant cells called
endospores
– Can survive heat that would rapidly kill vegetative
cells

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26.1 Heat Sterilization

• The autoclave is a sealed device that uses

steam under pressure (Figure 26.3)
– Allows temperature of water to get above 100C
– Not the pressure that kills things, but the high
temperature
• Pasteurization is the process of using precisely
controlled heat to reduce the microbial load in
heat-sensitive liquids
– Does not kill all organisms, so it is different than
sterilization

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Figure 26.3
Chamber
pressure
gauge
Steam exhaust
Steam
exhaust
valve

Door Jacket chamber

Thermometer
and valve Air exits through vent

Steam supply
valve
Steam enters here

Autoclave time
Stop
steam
Temperature (C)

Begin Sterilization time

pressure
Flowing Temperature Temperature
steam of object being of autoclave
sterilized

Total cycle time (min)

© 2012 Pearson Education, Inc.

26.2 Radiation Sterilization
• Microwaves, UV, X-rays, gamma rays, and
electrons can reduce microbial growth
• UV has sufficient energy to cause modifications
and breaks in DNA
– UV is useful for decontamination of surfaces
– Cannot penetrate solid, opaque, or light-absorbing
surfaces

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Figure 26.4

© 2012 Pearson Education, Inc.

26.2 Radiation Sterilization
• Ionizing radiation
– Electromagnetic radiation that produce ions and
other reactive molecules
– Generates electrons, hydroxyl radicals, and
hydride radicals
– Some microorganisms are more resistant to
radiation than others

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26.2 Radiation Sterilization
• Sources of radiation include cathode ray tubes,
X-rays, and radioactive nuclides
• Radiation is used for sterilization in the medical
field and food industry
– Radiation is approved by the WHO and is used in
the USA for decontamination of foods particularly
susceptible to microbial contamination
• Hamburger, chicken, spices may all be irradiated

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26.3 Filter Sterilization
• Filtration avoids the use of heat on sensitive
liquids and gases
– Pores of filter are too small for organisms to pass
through
– Pores allow liquid or gas to pass through
• Depth filters
– HEPA filters
– Membrane filters
– Function more like a sieve

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Figure 26.6

© 2012 Pearson Education, Inc.

26.3 Filter Sterilization
• Membrane filters (cont’d)
– Filtration can be accomplished by syringe, pump,
or vacuum
– A type of membrane filter is the nucleation track
(nucleopore) filter

© 2012 Pearson Education, Inc.

Figure 26.7

© 2012 Pearson Education, Inc.

Figure 26.8

© 2012 Pearson Education, Inc.

26.4 Chemical Growth Control
• Antimicrobial agents can be classified as
bacteriostatic, bacteriocidal, and bacteriolytic

© 2012 Pearson Education, Inc.

Figure 26.9

Bacteriostatic Bacteriocidal

Log cell number

Log cell number
Total cell count Total cell count

Viable cell count

Viable
cell count

Time Time

Bacteriolytic
Log cell number

Total cell count

Viable
cell count

Time

© 2012 Pearson Education, Inc.

26.4 Chemical Growth Control
• Minimum inhibitory concentration (MIC) is the
smallest amount of an agent needed to inhibit
growth of a microorganism
– Varies with the organism used, inoculum size,
temp, pH, etc.
• Disc diffusion assay
– Antimicrobial agent added to filter paper disc
– MIC is reached at some distance
• Zone of inhibition
– Area of no growth around disc

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Figure 26.10

Minimum
inhibitory
concentration

© 2012 Pearson Education, Inc.

Figure 26.11

Nutrient Inoculate plate

agar plate with a liquid
culture of a test
organism

Discs containing
antimicrobial
agents are placed
on surface

Incubate for 24–48 h

Test organism shows

susceptibility to some
agents, indicated by
inhibition of bacterial
growth around discs
(zones of inhibition)
© 2012 Pearson Education, Inc.
26.5 Chemical Antimicrobial Agents for
External Use
• These antimicrobial agents can be divided into two
categories
– Products used to control microorganisms in
commercial and industrial applications
• Examples: chemicals in foods, air-conditioning
cooling towers, textile and paper products, fuel
tanks
– Products designed to prevent growth of human
pathogens in inanimate environments and on
external body surfaces
• Sterilants, disinfectants, sanitizers, and antiseptics
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III. Antimicrobial Agents Used In Vivo
• Antimicrobial drugs are classified on the basis of
– Molecular structure
– Mechanism of action
– Spectrum of antimicrobial activity

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Figure 26.12

Cell wall synthesis DNA gyrase DNA-directed RNA polymerase

RNA elongation
Cycloserine Quinolones Nalidixic acid Rifampin
Vancomycin Ciprofloxacin Actinomycin
Streptovaricins
Bacitracin Novobiocin
Penicillins
Cephalosporins Protein synthesis
Monobactams
Carbapenems
(50S inhibitors)
Erythromycin (macrolides)
DNA Chloramphenicol
Clindamycin
Folic acid metabolism Lincomycin
Trimethoprim THF mRNA
Sulfonamides
Protein synthesis
Ribosomes (30S inhibitors)
DHF
50 50 50
Tetracyclines
30 30 30 Spectinomycin
Streptomycin
Gentamicin
Cytoplasmic membrane Kanamycin
structure and function Amikacin
Polymyxins Nitrofurans
Lipid
Daptomycin biosynthesis
Protein synthesis
Platensimycin (tRNA)
PABA Cytoplasmic Cell wall
membrane Mupirocin
Puromycin

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Figure 26.13

Eukaryotes Bacteria Obligately parasitic Bacteria Viruses

RNA DNA
Fungi Mycobacteria Gram-negative Gram-positive Chlamydia Rickettsia viruses viruses
Bacteria Bacteria

Tobramycin Penicillins
Nonnucleoside
Azoles reverse transcriptase
Allylamines Sulfonamides
inhibitors
Cycloheximide Cephalosporins
Protease inhibitors
Polyenes Streptomycin Quinolones
Fusion inhibitors
Polyoxins Tetracycline
Nucleic acid Nucleoside analogs
analogs Vancomycin Interferon
Isoniazid Polymyxins
Echinocandins Daptomycin

Platensimycin

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26.6 Synthetic Antimicrobial Drugs
• Paul Ehrlich studied selective toxicity in the early
1900s
– Selective toxicity is ability to inhibit or kill a
pathogen without affecting the host

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26.6 Synthetic Antimicrobial Drugs
• Sulfa drugs: discovered by Gerhard Domagk
in the 1930s
– Inhibit growth of bacteria (sulfanilamide is the
simplest;
– Isoniazid is a growth analog effective only
against Mycobacterium
• Interferes with synthesis of mycolic acid

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Figure 26.16

Sulfanilamide p-Aminobenzoic acid

Folic acid
© 2012 Pearson Education, Inc.
26.6 Synthetic Antimicrobial Drugs
• Nucleic acid base analogs have been formed by
the addition of bromine or fluorine
• Quinolones are antibacterial compounds that
interfere with DNA gyrase (e.g., ciprofloxacin)

© 2012 Pearson Education, Inc.

Figure 26.17
Growth factor Analog

Phenylalanine
(an amino acid) p-Fluorophenylalanine

Uracil 5-Fluorouracil
(an RNA base)

Thymine 5-Bromouracil
(a DNA base)
© 2012 Pearson Education, Inc.
26.7 Naturally Occurring Antimicrobial
Drugs: Antibiotics
• Antibiotics are naturally produced antimicrobial
agents
– Less than 1% of known antibiotics are clinically
useful
• Can be modified to enhance efficacy (semisynthetic)
• The susceptibility of microbes to different antibiotics
varies greatly
– Gram-positive and gram-negative bacteria vary in
their sensitivity to antibiotics
– Broad-spectrum antibiotics are effective against both
groups of bacteria
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26.8 -Lactam Antibiotics: Penicillins
and Cephalosporins
• -Lactam antibiotics are one of the most important
groups of antibiotics of all time
– Include penicillins, cephalosporins, and
cephamycins
– Over half of all antibiotics used worldwide
• Penicillins (Figure 26.19)
– Discovered by Alexander Fleming
– Primarily effective against gram-positive bacteria
– Some synthetic forms are effective against some
gram-negative bacteria
– Target cell wall synthesis
© 2012 Pearson Education, Inc.
Figure 26.19
N-Acyl group

-Lactam Thiazolidine
ring ring

6-Aminopenicillanic acid

N-Acyl group Designation

NATURAL PENICILLIN
Benzylpenicillin
(penicillin G)
Gram-positive activity
-lactamase-sensitive

SEMISYNTHETIC PENICILLINS

Methicillin
acid-stable,
-lactamase-resistant

Oxacillin
acid-stable,
-lactamase-resistant

Ampicillin
broadened spectrum of activity
(especially against gram-negative
Bacteria), acid-stable,
-lactamase-sensitive

Carbenicillin
broadened spectrum of activity
(especially against Pseudomonas
aeruginosa), acid-stable but
ineffective orally,
-lactamase-sensitive

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26.8 -Lactam Antibiotics: Penicillins
and Cephalosporins
• Cephalosporins (Figure 26.20)
– Produced by fungus Cephalosporium
– Same mode of action as the penicillins
– Commonly used to treat gonorrhea

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Figure 26.20

Dihydrothiazine
ring

-Lactam
ring

© 2012 Pearson Education, Inc.

26.9 Antibiotics from Prokaryotes
• Many antibiotics effective against Bacteria are
also produced by Bacteria
– Aminoglycosides are antibiotics that contain
amino sugars bonded by glycosidic linkage
(Figure 26.21)
• Examples: kanamycin, neomycin, amikacin
– Not commonly used today
• Neurotoxicity and nephrotoxicity
• Considered reserve antibiotics for when other
antibiotics fail

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Figure 26.21

N-Acetyltransferase

Streptomycin Kanamycin
© 2012 Pearson Education, Inc.
26.9 Antibiotics from Prokaryotes
• Macrolides contain lactone rings bonded to
sugars (Figure 26.22)
– Example: erythromycin
– Broad-spectrum antibiotic that targets the 50S
subunit of ribosome
• Tetracyclines contain four rings (Figure 26.23)
– Widespread medical use in humans and animals
– Broad-spectrum inhibition of protein synthesis
– Inhibits functioning of 30S ribsomal subunit

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Figure 26.22

Macrolide
ring

Sugars

© 2012 Pearson Education, Inc.

Figure 26.23

Tetracycline analog R1 R2 R3 R4

Tetracycline H OH CH3 H

7-Chlortetracycline H OH CH3 Cl
(aureomycin)

5-Oxytetracycline OH OH CH3 H
(terramycin)
© 2012 Pearson Education, Inc.
26.9 Antibiotics from Prokaryotes
• Daptomycin (Figure 26.24)
– Also produced by Streptomyces
– Used to treat gram-positive bacterial infections
– Forms pores in cytoplasmic membrane
• Platensimycin
– New structural class of antibiotic (Figure 26.25)
– Broad-spectrum, effective against MRSA and
vancomycin-resistant enterococci

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Figure 26.24

© 2012 Pearson Education, Inc.

Figure 26.25

© 2012 Pearson Education, Inc.

26.10 Antiviral Drugs
• Most antiviral drugs also target host structures,
resulting in toxicity
• Most successful and commonly used antivirals
are the nucleoside analogs (e.g., AZT)
– Block reverse transcriptase and production of
viral DNA
– Also called nucleoside reverse transcriptase
inhibitors
• Nonnucleoside reverse transcriptase inhibitors
(NNRTI) bind directly to RT and inhibit reverse
transcription
© 2012 Pearson Education, Inc.
26.10 Antiviral Drugs
• Protease inhibitors inhibit the processing of large
viral proteins into individual components
• Fusion inhibitors prevent viruses from successfully
fusing with the host cell
• Two categories of drugs successfully limit
influenza infection:
– Adamantanes
– Neuraminidase inhibitors
• Interferons are small proteins that prevent viral
multiplication by stimulating antiviral proteins in
uninfected cells
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26.11 Antifungal Drugs
• Fungi pose special problems for chemotherapy
because they are eukaryotic (Figure 26.26)
– Much of the cellular machinery is the same as
that of animals and humans
– As a result, many antifungals are topical
– A few drugs target unique metabolic processes
unique to fungi

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26.12 Antimicrobial Drug Resistance
• Antimicrobial drug resistance
– The acquired ability of a microorganism to
resist the effects of a chemotherapeutic agent
to which it is normally sensitive

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26.12 Antimicrobial Drug Resistance
• Most drug-resistant bacteria isolated from
patients contain drug-resistance genes located
on R plasmids
• Evidence indicates that R plasmids predate the
antibiotic era
• The use of antibiotics in medicine, veterinary
medicine, and agriculture selects for the spread
of R plasmids (Figure 26.28)
– Many examples of overuse of antibiotics
– Used far more often than necessary
(e.g., antibiotics used in agriculture as
supplements to animal feed)
© 2012 Pearson Education, Inc.
26.12 Antimicrobial Drug Resistance
• Almost all pathogenic microbes have acquired
resistance to some chemotherapeutic agents
(Figure 26.29)
• A few pathogens have developed resistance to all
known antimicrobial agents
– Methicillin-resistant S. aureus (MRSA)
• Resistance can be minimized by using antibiotics
correctly and only when needed
• Resistance to a certain antibiotic can be lost if
antibiotic is not used for several years

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Figure 26.29
Candida albicans
Acinetobacter spp.
Gram-negative Enterococcus faecalis*
Gram-positive
Streptococcus pneumoniae
Gram-positive/
acid-fast Mycobacterium tuberculosis*
Fungus
Haemophilus ducreyi
Salmonella typhi
Haemophilus influenzae
Neisseria gonorrhoeae
Pseudomonas aeruginosa*
Salmonella spp.
Shigella dysenteriae
Shigella spp.
Other gram-negative rods
Staphylococcus aureus

Year
© 2012 Pearson Education, Inc.
26.13 The Search for New Antimicrobial
Drugs
• Long-term solution to antimicrobial resistance
relies on the development of new antimicrobial
compounds
– Modification of current antimicrobial compounds
is often productive
– Automated chemistry methods (combinatorial
chemistry) has sped up drug discovery
– 7,000,000 compounds must be screened to find
a single useful clinical drug

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26.13 The Search for New Antimicrobial
Drugs
• Computers can now be used to design molecules to
interact with specific microbial structures
– Most successful example is saquinavir
• Binds to active site of HIV protease
• New methods of screening natural products are
being used
– Led to the discovery of platensimycin
• Combinations of drugs can be used (e.g., ampicillin
and sulbactam)
• Bacteriophage therapy
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