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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 100C– 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 26.3Chamberpressuregauge
Steamexhaustvalve
Door
Thermometerand valve
Steam supplyvalve
Steam enters here
Steam exhaust
Jacket chamber
Air exits through vent
Total cycle time (min)
Tem
per
atu
re (
C)
Autoclave time
Stop steam
Beginpressure
Flowingsteam
Sterilization time
Temperatureof object beingsterilized
Temperatureof autoclave
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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
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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
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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
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Figure 26.7
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Figure 26.8
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26.4 Chemical Growth Control
• Antimicrobial agents can be classified as bacteriostatic, bacteriocidal, and bacteriolytic
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Figure 26.9
Total cell count
Viable cell count
Time
Lo
g c
ell n
um
ber
Lo
g c
ell n
um
ber
Lo
g c
ell n
um
ber
Bacteriostatic Bacteriocidal
Bacteriolytic
Total cell count
Total cell count
Time
Time
Viable cell count
Viable cell count
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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
Minimuminhibitoryconcentration
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Figure 26.11
Nutrientagar plate
Discs containingantimicrobialagents are placedon surface
Inoculate platewith a liquidculture of a testorganism
Incubate for 24–48 h
Test organism showssusceptibility to someagents, indicated byinhibition of bacterialgrowth around discs(zones of inhibition)
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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
CycloserineVancomycinBacitracinPenicillinsCephalosporinsMonobactamsCarbapenems
TrimethoprimSulfonamides
Quinolones
Cell wall synthesis
Folic acid metabolism
DNA gyrase
Nalidixic acidCiprofloxacinNovobiocin
Cytoplasmic membranestructure and function
PolymyxinsDaptomycin
THF
DHF
DNA
mRNA
Ribosomes
50 50 50
30 30 30
RNA elongation
Actinomycin
DNA-directed RNA polymerase
RifampinStreptovaricins
Protein synthesis(50S inhibitors)
Erythromycin (macrolides)ChloramphenicolClindamycinLincomycin
Protein synthesis(30S inhibitors)
TetracyclinesSpectinomycinStreptomycinGentamicinKanamycinAmikacinNitrofurans
Protein synthesis(tRNA)
Lipidbiosynthesis
MupirocinPuromycin
PlatensimycinCell wallCytoplasmic
membrane
PABA
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Figure 26.13
Fungi
Eukaryotes Bacteria Obligately parasitic Bacteria Viruses
AzolesAllylamines
CycloheximidePolyenesPolyoxins
Nucleic acidanalogs
Echinocandins
Mycobacteria Gram-negativeBacteria
Gram-positiveBacteria
Tobramycin Penicillins
Streptomycin
SulfonamidesCephalosporins
Quinolones
Isoniazid Polymyxins
Tetracycline
VancomycinDaptomycin
Platensimycin
Chlamydia RickettsiaRNA
virusesDNA
viruses
Nonnucleosidereverse transcriptase
inhibitorsProtease inhibitorsFusion inhibitors
Nucleoside analogsInterferon
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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
Folic acid
p-Aminobenzoic acid
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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)
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Figure 26.17 Growth factor Analog
Phenylalanine(an amino acid) p-Fluorophenylalanine
5-Fluorouracil
5-Bromouracil
Uracil(an RNA base)
Thymine(a DNA base)
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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
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Figure 26.19
N-Acyl group
-Lactamring
Thiazolidinering
6-Aminopenicillanic acid
N-Acyl group Designation
NATURAL PENICILLIN
SEMISYNTHETIC PENICILLINS
Benzylpenicillin(penicillin G)
Methicillin
Oxacillin
Ampicillin
Carbenicillin
Gram-positive activity-lactamase-sensitive
acid-stable,-lactamase-resistant
acid-stable,-lactamase-resistant
broadened spectrum of activity(especially against gram-negativeBacteria), acid-stable,-lactamase-sensitive
broadened spectrum of activity(especially against Pseudomonasaeruginosa), acid-stable butineffective 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
Dihydrothiazinering
-Lactamring
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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
Streptomycin Kanamycin
N-Acetyltransferase
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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
Macrolidering
Sugars
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Figure 26.23
Tetracycline analog R1 R2 R3 R4
Tetracycline
7-Chlortetracycline (aureomycin)
5-Oxytetracycline (terramycin)
H
H
HOH
OH
OHOH H
Cl
CH3
CH3
CH3
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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
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Figure 26.25
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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
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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)
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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
Gram-negativeGram-positive
Gram-positive/acid-fastFungus
Other gram-negative rods
Year
Candida albicans
Acinetobacter spp.
Enterococcus faecalis*
Streptococcus pneumoniae
Mycobacterium tuberculosis*
Haemophilus ducreyi
Salmonella typhi
Haemophilus influenzae
Neisseria gonorrhoeae
Pseudomonas aeruginosa*
Salmonella spp.
Shigella dysenteriae
Shigella spp.
Staphylococcus aureus
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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© 2012 Pearson Education, Inc.