8 and 9
Control of
Microorganisms in the
Environment and
Antimicrobial
Chemotherapy
1
Copyright © McGraw-Hill Global Education Holdings, LLC. Permission required for reproduction or display.
2
Definition of Frequently Used
Terms
• Sterilization
– destruction or removal of all viable organisms
• Disinfection
– killing, inhibition, or removal of disease
causing (pathogenic) organisms
– disinfectants
• agents, usually chemical, used for disinfection
• usually used on inanimate objects
3
More Definitions…
• Sanitization
– reduction of microbial population to levels
deemed safe (based on public health
standards)
• Antisepsis
– prevention of infection of living tissue by
microorganisms
– antiseptics
• chemical agents that kill or inhibit growth of
microorganisms when applied to tissue
4
Antimicrobial Agents
• Chemotherapy
– use of chemicals to kill or inhibit growth of
microorganisms within host tissue
• Agents that kill microorganisms or inhibit their
growth
– cidal agents kill
– static agents inhibit growth
5
-cidal vs. –static Agents
-cide
– suffix indicating that agent kills
– germicide
• kills pathogens and many nonpathogens but not
necessarily endospores
– include bactericides, fungicides, algicides, and
viricides
-static
– suffix indicating that agent inhibits growth
– include bacteriostatic and fungistatic
6
The Pattern of Microbial Death
• Microorganisms are not killed instantly
• Population death usually occurs exponentially
• Measure of agent’s killing efficiency
– decimal reduction time (D-value) – time to
kill 90%
– must be sure persister cells (viable but
nonculturable (VBNC) condition) are dead
• once they recover they may regain the ability to
reproduce and cause infection
7
8
9
Conditions Influencing the
Effectiveness of Antimicrobial Agent
Activity
• Population size
– larger populations take longer to kill than
smaller populations
• Population composition
– microorganisms differ markedly in their
sensitivity to antimicrobial agents
10
More Conditions…
• Concentration or intensity of an antimicrobial agent
– usually higher concentrations kill more rapidly
– relationship is not linear
• Duration of exposure
longer exposure ⇒ more organisms killed
• Temperature
– higher temperatures usually increase killing
• Local environment
– pH, viscosity, concentration of organic matter, etc.
can profoundly impact effectiveness
– organisms in biofilms are less susceptible to many
antimicrobial agents
11
Filtration
• Reduces microbial population or sterilizes
solutions of heat-sensitive materials by
removing microorganisms
• Also used to reduce microbial populations in
air
12
Filtering Liquids
• Membrane filters
– porous membranes with defined pore sizes that
remove microorganisms primarily by physical
screening
13
Filtering Air
• Surgical masks
• Cotton plugs on
culture vessels
• High-efficiency
particulate air
(HEPA) filters
– used in laminar
flow biological
safety cabinets
14
Physical Control Methods
• Heat
–
–
–
–
–
Moist heat
Steam: Autoclave
Pasteurization
Dry heat
Incineration
• Radiation
– Ultraviolet (UV)
– Ionizing (gamma)
15
Moist Heat
• Destroys viruses, fungi, and bacteria
• Boiling will not destroy spores and does not sterilize
• Degrades nucleic acids, denatures proteins,
and disrupts membranes
16
Steam Sterilization
• Carried out above
100oC which requires
saturated steam under
pressure
• Uses an autoclave
• Effective against all
types of
microorganisms
(including spores!)
• Quality control includes strips with
Geobacillus
stearothermophilus
17
Pasteurization
• Controlled heating at temperatures well below
boiling
• Used for milk, beer, and other beverages
• Process does not sterilize but does kill
pathogens present and slow spoilage by
reducing the total load of organisms present
18
Dry Heat Sterilization
• Less effective than moist heat sterilization,
requiring higher temperatures and longer
exposure times
– items subjected to 160–170oC for 2 to 3 hours
• Oxidizes cell constituents and denatures
proteins
19
Dry Heat Incineration
• Bench top incinerators are used to sterilize
inoculating loops used in microbiology
laboratories
20
Ultraviolet (UV) Radiation
• Wavelength of 260 is most bactericidal (DNA
absorbs)
• Causes thymine dimers preventing
replication and transcription
• UV limited to surface sterilization because it
does not penetrate glass, dirt films, water,
and other substances
• Has been used for water treatment
21
Ionizing Radiation
• Gamma radiation penetrates deep into objects and
forms reactive high energy radicals which are
destructive to the DNA and proteins.
• Destroys bacterial endospores; not always effective
against viruses
• Used for sterilization and pasteurization of
antibiotics, hormones, sutures, plastic disposable
supplies, and food
22
Chemical Control Agents
• Disinfection
• Antisepsis
• Sterilization
23
Chemical Agents
should be • Effective against wide variety of infectious
agents
• Selectively toxic
• Effective at low concentrations
• Effective in the presence of organic matter
• stable in storage
• homogeneous
Overuse of antiseptics such as triclosan has
selected for triclosan resistant bacteria and
possibly antibiotic resistant
24
25
26
Phenolics
• Commonly used as laboratory and hospital
disinfectants
• Act by denaturing proteins and disrupting cell
membranes
• Tuberculocidal, effective in presence of organic
material, and long lasting
• Disagreeable odor and can cause skin irritation
27
Alcohols
• Among the most widely used disinfectants and
antiseptics
• Two most common are ethanol and isopropanol
• Bactericidal, fungicidal, but not sporicidal
• Inactivate some viruses
• Denature proteins and possibly dissolve
membrane lipids
28
Halogens
• Any of five elements: fluorine, chlorine,
bromine, iodine, and astatine
• Important antimicrobial agents
29
Halogens - Iodine
• Skin antiseptic
• Oxidizes cell constituents and iodinates
proteins
• At high concentrations may kill spores
• Skin damage, staining, and allergies can be a
problem
• Iodophore
– iodine complexed with organic carrier
– released slowly to minimize skin burns
30
Halogens - Chlorine
• Oxidizes cell constituents
• Important in disinfection of water supplies and
swimming pools, used in dairy and food
industries, effective household disinfectant
• Destroys vegetative bacteria and fungi,
• Chlorine gas is sporicidal
• Can react with organic matter to form
carcinogenic compounds – THMs
(trihalomethanes)
31
Heavy Metals
• e.g., ions of mercury, silver, arsenic, zinc, and
copper
• Effective but usually toxic
• Combine with and inactivate proteins; may
also precipitate proteins
32
Quaternary Ammonium
Compounds
• detergents that have antimicrobial activity and are
effective disinfectants
– amphipathic organic cleansing agents
• most likely disrupts cell membrane
• cationic detergents are effective disinfectants
– kill most bacteria, but not M. tuberculosis or endospores
– safe and easy to use, inactivated by hard water and soap
33
Aldehydes
• Commonly used agents are formaldehyde
and glutaraldehyde
• Highly reactive molecules
• Sporicidal and can be used as chemical
sterilants
• Combine with and inactivate nucleic acids
and proteins
34
Sterilizing Gases
• Used to sterilize heat-sensitive materials
• Microbicidal and sporicidal
• Ethylene oxide sterilization is carried out in
equipment resembling an autoclave
• Betapropiolactone and vaporized hydrogen
peroxide
• Combine with and inactivate DNA and proteins
35
Evaluation of Antimicrobial
Agent Effectiveness
• Complex process regulated by U.S. federal
agencies
– Environmental Protection Agency
– Food and Drug Administration
36
Phenol Coefficient Test
• Potency of a disinfectant is compared to that
of phenol
• Useful for screening but may be misleading
– Phenol has a residual effectiveness
– Protocol involves adding disinfectant and
organism to the same tube which is different
than how disinfectants are really used
37
Other Evaluation Methods
• Use dilution test
– determines rate at which selected bacteria are
destroyed by various chemical agents
– Protocol involves contaminating a bead and
then disinfecting
• Normal in-use testing
– testing done using conditions that approximate
normal use of disinfectant
38
Biological Control of
Microorganisms
• Emerging field showing great promise
• Natural control mechanisms
– predation by Bdellovibrio
– viral-mediated lysis using pathogen specific
bacteriophage, currently limited use in food
industry but expanded
– toxin-mediated killing using bacteriocins
39
Chemotherapeutic Agents
• Chemical agents used to treat disease
• Destroy pathogenic microbes or inhibit their
growth within host
• Most are antibiotics
– microbial products or their derivatives that kill
susceptible microbes or inhibit their growth
40
The Development of Chemotherapy
• Paul Ehrlich (1904)
– developed concept of selective toxicity
– identified dyes that effectively treated African
sleeping sickness
• Sahachiro Hato (1910)
– working with Ehrlich, identified arsenic
compounds that effectively treated syphilis
• Gerhard Domagk, and Jacques and Therese
Trefouel (1935)
– discovered sulfonamides and sulfa drugs
41
Penicillin
• First discovered by Ernest Duchesne (1896),
but discovery lost
• Accidentally discovered by Alexander
Fleming (1928)
– observed penicillin activity on contaminated
plate
– did not think could be developed further
• Effectiveness demonstrated by Florey, Chain,
and Heatley (1939)
• Fleming, Florey, and Chain received Nobel
Prize in 1945 for discovery and production of
penicillin
42
43
Later Discoveries
• Streptomycin, an antibiotic active against
tuberculosis, was discovered by Selman
Waksman (1944)
– Nobel Prize was awarded to Waksman in
1952 for this discovery
• By 1953 chloramphenicol, terramycin,
neomycin, and tetracycline isolated
44
General Characteristics of
Antimicrobial Drugs
• Selective toxicity
– ability of drug to kill or inhibit pathogen while
damaging host as little as possible
• Therapeutic dose
– drug level required for clinical treatment
• Toxic dose
– drug level at which drug becomes too toxic for
patient (i.e., produces side effects)
• Therapeutic index (high TI is best)
– ratio of toxic dose to therapeutic dose
– Minimum toxic dose to the host/minimum effective
microbial lethal dose
45
General Characteristics of
Antimicrobial Drugs…
• Side effects – undesirable effects of drugs on
host cells
• Narrow-spectrum drugs – attack only a few
different pathogens
• Broad-spectrum drugs – attack many different
pathogens
• Cidal agent - kills microbes
• Static agent - inhibits growth of microbes
46
47
48
General Characteristics of
Antimicrobial Drugs…
• Effect of an agent may vary
– with concentration, microbe, host
• Effectiveness expressed in two ways
– minimal inhibitory concentration (MIC)
• lowest concentration of drug that inhibits growth
of pathogen
– minimal lethal concentration (MLC)
• lowest concentration of drug that kills pathogen
49
Kirby-Bauer Method
• Standardized method for disk diffusion test
• Sensitivity/resistance determined using tables
relating zone diameter with microbial resistance
• Table values plotted, used to determine if effective
concentration of drug in body can be reached
50
The E Test
• Convenient for use with
anaerobic pathogens
• Similar to disk diffusion
method, but uses strip
rather than disk
• E-test strips contain a
gradient of an antibiotic
• Intersection of elliptical
zone of inhibition with
strip indicates MIC
51
Antimicrobial Drugs
Classified By Their
Mode of Action:
1. Inhibitors of cell wall synthesis
2. Protein synthesis inhibitors
3. Metabolic antagonists
4. Nucleic acid synthesis inhibition
52
1. Inhibitors of Cell Wall
Synthesis
• Penicillins
– most are 6-aminopenicillanic acid derivatives
and differ in side chain attached to amino
group
– most crucial feature of molecule is the βlactam ring
• essential for bioactivity
• many penicillin resistant organisms produce βlactamase (penicillinase) which hydrolyzes a
bond in this ring
53
Penicillins…
• Mode of action
– blocks the enzyme that
catalyzes
transpeptidation
(formation of cross-links
in peptidoglycan)
– prevents the synthesis
of complete cell walls
leading to lysis of cell
– acts only on growing
bacteria that are
synthesizing new
peptidoglycan
54
Other Actions of Penicillins
• Binds to periplasmic proteins (penicillinbinding proteins, PBPs)
• May activate bacterial autolysins and murein
hydrolases
• Stimulate bacterial holins to form holes or
lesions in the plasma membrane
55
Penicillins…
• Naturally occurring penicillins
– penicillin V and G are narrow spectrum
• Semisynthetic penicillins have a broader
spectrum than naturally occurring ones
• Resistance to penicillins, including the
semisynthetic analogs, continues to be a
problem
• ~1–5% of adults in U.S. are allergic to
penicillin
– allergy can lead to a violent allergic response
and death
56
Cephalosporins
• Structurally and functionally similar to penicillins
• Broad-spectrum antibiotics that can be used by
most patients that are allergic to penicillin
• Four categories based on their spectrum of activity
57
Vancomycin and Teicoplanin
• Glycopeptide antibiotics
• Inhibit cell wall synthesis
• Vancomycin - important
for treatment of antibioticresistant staphylococcal
and enterococcal
infections
– previously considered
“drug of last resort” so
rise in resistance to
vancomycin is of great
concern
58
2. Protein Synthesis Inhibitors
• Many antibiotics bind specifically to the
bacterial ribosome
– binding can be to 30S (small) or 50S (large)
ribosomal subunit
• Other antibiotics inhibit a step in protein
synthesis
–
–
–
–
aminoacyl-tRNA binding
peptide bond formation
mRNA reading
translocation
59
Aminoglycoside Antibiotics
• Large group, all with a cyclohexane ring, amino sugars
• Neomycin, gentamicin, streptomycin, tobramycin
• Bind to 30S ribosomal subunit, interfere with protein
synthesis by directly inhibiting the process and by
causing misreading of the messenger RNA
• Resistance and toxicity
60
Tetracyclines
• All have a four-ring structure to which a variety of
side chains are attached
• Are broad spectrum, bacteriostatic
• Combine with 30S ribosomal subunit
– inhibits bind of aminoacyl-tRNA molecules to the A
site of the ribosome
• Sometimes used to treat acne
61
Macrolides
• Contain 12- to 22-carbon
lactone rings linked to one
or more sugars
• e.g., erythromycin
– broad spectrum, usually
bacteriostatic
– binds to 23S rRNA of 50S
ribosomal subunit
• inhibits peptide chain
elongation
• Used for patients allergic
to penicillin
62
Chloramphenicol
• Now is chemically synthesized
• Binds to 23s rRNA on 50S ribosomal subunit
and inhibits peptidyl transferase reaction
• Toxic with numerous side effects so only
used in life-threatening situations
63
3. Metabolic Antagonists
• Act as antimetabolites
– antagonize or block functioning of metabolic
pathways by competitively inhibiting the use of
metabolites by key enzymes
• Are structural analogs
– molecules that are structurally similar to, and
compete with, naturally occurring metabolic
intermediates
• block normal cellular metabolism
64
Sulfonamides or Sulfa Drugs
• Structurally related to
sulfanilamide, a
paminobenzoic acid
(PABA) analog
• PABA used for the
synthesis of folic acid
and is made by many
pathogens
– sulfa drugs are
selectively toxic due to
competitive inhibition
of folic acid synthesis
enzymes
65
Trimethoprim
• Synthetic antibiotic that also interferes with
folic acid production
• Broad spectrum
• Can be combined with sulfa drugs to increase
efficacy of treatment: Bactrim or Septra
– combination blocks two steps in folic acid
pathway
• Has a variety of side effects including
abdominal pain and photosensitivity reactions
66
Nucleic Acid Synthesis Inhibition
• A variety of mechanisms
– block DNA replication
• inhibition of DNA polymerase
• inhibition of DNA helicase
– block transcription
• inhibition of RNA polymerase
• Drugs not as selectively toxic as other
antibiotics because bacteria and eukaryotes
do not differ greatly in the way they
synthesize nucleic acids
67
Quinolones
• Broad-spectrum, synthetic drugs containing the 4quinolone ring: Cipro (ciprofloxacin) and Levaquin
• Nalidixic acid first synthesized quinolone (1962)
• Act by inhibiting bacterial DNA-gyrase and
topoisomerase II
• Broad spectrum, bactericidal, wide range of
infections
68
Factors Influencing Antimicrobial
Drugs
• Ability of drug to reach site of infection
• Susceptibility of pathogen to drug
• Ability of drug to reach concentrations in body
that exceed MIC of pathogen
69
Ability of Drug to Reach Site of
Infection
• Depends in part on mode of administration
– oral
• some drugs destroyed by stomach acid
– topical
– parenteral routes
• nonoral routes of administration
• Drug can be excluded by blood clots or
necrotic tissue
70
Factors Influencing Ability of
Drug to Reach Concentrations
Exceeding MIC
• Amount administered
• Route of administration
• Speed of uptake
• Rate of clearance (elimination) from body
71
Drug Resistance
• An increasing problem
– once resistance originates in a population it
can be transmitted to other bacteria
– a particular type of resistance mechanism is
not confirmed to a single class of drugs
• Microbes in abscesses or biofilms may be
growing slowly and not be susceptible
• Resistance mutants arise spontaneously and
are then selected
72
Overcoming Drug Resistance
• Give drug in appropriate concentrations to
destroy susceptible
• Give two or more drugs at same time
• Use drugs only when necessary
• Possible future solutions
– continued development of new drugs
– use of bacteriophages to treat bacterial
disease
73