Transcript Lecture 21

Chapter 7 and 20
The Control of Microbial Growth
Antimicrobial Drugs
The Control of Microbial Growth -Terminology
• Sterilization: Removal of all microbial life
• Sanitization: Lower microbial counts, reduce the number of
microorganisms to a safe level ( eating utensils)
• Disinfection: Removal of pathogens (applied to non-living objects)
• Degerming: Removal of microbes from a limited area of living
tissue (the skin)
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Sepsis refers to microbial contamination.
Asepsis is the absence of significant contamination.
Antisepsis: Removal of pathogens from living tissue
Aseptic surgery techniques prevent microbial contamination of
wounds.
Actions of Microbial Control Agents
• Biocide/Germicide: Kills microbes
• Bacteriostasis: Inhibiting, not killing, microbes
• Alteration of membrane permeability
• Damage to proteins
• Damage to nucleic acids
Figure 7.1b
The Rate of Microbial Death
Bacterial populations die at a constant logarithmic rate.
Figure 7.1a
Table 7.2 Microbial Exponential Death Rate: An Example
Effectiveness of antimicrobial treatment
Depends on:
1. Number of microbes
2. Environment
• The presence of organic matter
• Temperature - increased temperatures usually enhance the efficacy of
antimicrobial treatment
– Time of exposure
– Microbial characteristics
• resistance
• capsules
• biofilms
Physical Methods of Microbial Control
1. Heat
– Thermal death point (TDP) – lowest temperature at which all the
microorganisms in a particular liquid suspension will be killed in 10 min
– Thermal death time (TDT): Time it takes to kill a specific bacteria at a specific
temperature.
• Dry Heat Sterilization kills by oxidation
– Hot air sterilization
– Direct flaming
• Moist Heat Sterilization - denatures proteins
• Boiling or flowing steam
• Kills bacteria, fungi and viruses
• Less effective on endospores
• Autoclave - steam under pressure - denatures proteins
• Kills endospores too
Equivalent treatments
Hot-air
Autoclave
170˚C, 2 hrs.
121˚C, 15 min
sterilization indicators
Physical Methods of Microbial Control
• Pasteurization reduces spoilage organisms and pathogens.
Equivalent treatments:
– 63°C for 30 min
– High-temperature short-time 72°C for 15 sec
– Ultra-high-temperature: 140°C for <1 sec
Physical Methods of Microbial Control
2. Filtration
– Sterilization of liquids thru membrane with pore size 0.2 µm removes
microbes
• Enzymes
• Vaccines
– High-efficiency particulate air (HEPA) filters
• removes microbes >0.3 µm
• Lower the numbers of airborne microbes
3. Low temperature inhibits microbial growth
– Refrigeration
– Deep freezing
– Lyophilization – freeze-drying
4. High pressure denatures proteins
5. Desiccation prevents metabolism
6. Osmotic pressure causes plasmolysis
Physical Methods of Microbial Control
7. Radiation damages DNA
– Ionizing radiation (X rays, gamma rays, electron
beams)
• Use in pharmaceuticals, medical and dental supplies
– Nonionizing radiation (UV)
• Control of closed environment
– Microwaves kill by heat; not especially antimicrobial
Chemical Methods of Microbial Control
• Antiseptics - chemical agents used on living tissue
• Disinfectants - chemical agents used on inanimate objects
• Levels of chemical decontamination
– High level germicides kill endospores, and if properly used, are
sterilants.
– Intermediate germicides kill fungal (not bacterial) spores, resistant
pathogens such as tubercle bacillus, and viruses (used for non-invasive
equipment).
– Low-level germicides kill vegetative bacteria and fungal cells, and
some viruses (used for materials that may touch the skin, not mucous
membrane).
Principles of effective chemical decontamination
• Properties of the disinfectant – how it works determines
what it will be effective against.
• Concentration of the disinfectant
• The degree of contact with microorganisms – if the surface
needs cleaning or is porous microorganisms can escape
contact with the disinfectant.
• Temperature should also be considered, since increased
temperatures usually enhance the efficacy of disinfectants.
• Time left in contact is also an important consideration.
Chemical antimicrobial agents
1. Halogens: non-metallic elements that commonly occur in minerals,
seawater, and salts
– Chlorine, hypochlorite (Clorox), chloramines, iodine, iodophors
– Oxidizing agents (Bleach is hypochlorous acid (HOCl))
• Denaturation of proteins.
• Can be sporicidal.
2. Phenol (Phenolics) – Lysol, Triclosan: soap antibacterial additive
– Damage cell membrane & precipitate proteins; bactericidal, fungicidal,
virucidal, not sporicidal.
– Active in organic matter.
3. Chlorhexidine - halogen and phenol compound
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Disrupt plasma membranes
Binds well to skin and mucus membranes
Low toxicity
Surgical hand scrubs and pre-op skin prep
Not sporicidal
Chemical antimicrobial agents
4. Alcohols - Ethanol, Isopropanol
– Denature proteins
– Dissolve lipids
– Denaturation requires H2O, which is
why aqueous preparations are better
than pure alcohols.
70% is best alcohol concentration
5. Hydrogen Peroxide
- Weak (3%) to strong (35%)
- Produce highly reactive hydroxyls: (free radicals) that damage protein
& DNA while also decomposing to O2 gas, toxic to anaerobes.
– Strong solutions are sporicidal
Table 7.6
Chemical antimicrobial agents
6. Detergents and soaps - Quaternary ammonium compounds
(QUATS) - surfactants that alter membrane proteins of some bacteria
and fungi.
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Not sporicidal
Hospital cleaners
Smells nice
Soaps: mechanically remove dirt/soil and grease containing microbes
7. Heavy Metals - Ag (Silver), Hg (mercury), Cu (copper), and Zn
(zinc) are used as germicidals.
– Oligodynamic action -When heavy metal ions combine with sulfhydryl (-SH)
groups, proteins are denatured.
– Not sporicidal.
Chemical antimicrobial agents
8. Aldehyde - glutaraldehyde and formaldehyde
– Kill everything by crosslinking/alkylating proteins & DNA.
– Glutaraldehyde in 2% solution (T.B.) used as sterilant for heat sensitive
instruments.
– Formaldehyde: disinfectant, preservative, toxicity limits use.
9. Gases and Aerosols - Ethylene oxide, propylene oxide,
betapropiolactone, and chlorine dioxide.
– Strong alkylating agents, sporicidal.
– Equipment, bedding, disposables, sterilization.
– Very toxic (eyes, skin, mucus, membranes). Carcinogenic?
10. Organic Acids - Used in foods to inhibit microbial growth.
– Sulfur dioxide, Sodium benzoate/benzoic acid, Sorbic acid, Propionic
acid/Ca propionate, Nitrates and Nitrites
Microbial Characteristics and Microbial Control
Figure 7.11
Evaluating a disinfectant
Evaluating a disinfectant
Disk-diffusion method
Figure 7.6
Learning objectives
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Define the following key terms related to microbial control: sterilization, disinfection,
antisepsis, degerming, sanitization, biocide, germicide, bacteriostasis, and asepsis.
Describe the patterns of microbial death caused by treatments with microbial control agents.
Describe the effects of microbial control agents on cellular structures.
Compare the effectiveness of moist heat (boiling, autoclaving, pasteurization) and dry heat.
Describe how filtration, low temperature, high pressure, desiccation, and osmotic pressure
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suppress microbial growth.
Explain how radiation kills cells.
List the factors related to effective disinfection.
Interpret results of disk-diffusion method.
Identify the methods of action and preferred uses of chemical disinfectants.
Identify the appropriate uses for surface-active agents.
Identify the method of sterilizing plastic lab ware.
Explain how microbial control is affected by the type of microbe.
Antimicrobial Drugs
• Chemotherapy - The use of drugs to treat a disease
• Antimicrobial drugs - A chemical substance that destroys
pathogenic microorganisms with minimal damage to host tissues
– Depending on the type of microbe the drug targets they can be
classified as antibacterial, antifungal, antiviral, or antiparasitic.
• Antibiotic - Substance produced by a microbe that, in small amounts,
inhibits another microbe
• Selective toxicity - A drug that kills harmful microbes without
damaging the host
The History of Chemotherapy
• Paul Ehrlich developed the concept of chemotherapy to treat
microbial diseases;
– He predicted the development of chemotherapeutic agents,
which would kill pathogens without harming the host.
• 1928 – Fleming discovered penicillin,
produced by Penicillium chrisogenes.
– 1940 – Howard Florey and Ernst Chain
performed first clinical trials of penicillin.
• Sulfa drugs came into prominence in the late 1930s.
Table 20.1
Antimicrobial drugs
• Spectrum of antimicrobial drugs
• The action of antimicrobial drugs
– General action is either by
• directly killing microorganisms (bactericidal)
• inhibiting their growth (bacteriostatic).
– Antibiotic activity
• MIC - Minimal inhibitory concentration
• MBC - Minimal bactericidal concentration
• Pharmacology
• Blood concentration
• Tissue distribution
• Adverse reactions
– Antimicrobial agents should not cause excessive harm to normal microbiota.
– Toxicity - Neuro, nephro and GI system
– Hypersensitivity
Figure 20.7
• A superinfections results from a disruption of normal microbiota
– when a pathogen develops resistance to the drug being used.
– when normally resistant microbiota multiply excessively.
The Spectrum of Antimicrobial drugs
• Antibacterial drugs affect different targets in a prokaryotic cell.
– Narrow-spectrum drugs affect only a select group of
microbes - gram-positive cells, for example;
– Broad-spectrum drugs affect a more diverse range of
microbes.
• Fungal, protozoan, and helminthic infections are more difficult
to treat because these organisms have eukaryotic cells.
Figure 20.2 Major Action Modes of Antimicrobial Drugs.
1. Inhibition of cell wall synthesis: penicillins,
2. Inhibition of protein synthesis: chloramphenicol,
cephalosporins, bacitracin, vancomycin
erythryomycin, tetracyclines, streptomycin
DNA
mRNA
Transcription
Protein
Translation
Replication
Enzyme
4. Injury to plasma
membrane:
polymyxin B
5. Inhibition of essential
metabolite synthesis:
sulfanimide, trimethoprim
3. Inhibition of nucleic acid replication
and transcription: quinolones, rifampin
Sites of action of antimicrobial drugs
1. Inhibition of cell wall synthesis
– can inhibit NAG and NAM synthesis or crosslinking
– peptidoglycan not found in humans
– only affects actively growing cells: new walls
– bacteriostatic
– gram negatives less susceptible
– Examples:
• Penicillin is the classic example of an inhibitor of cell wall synthesis
• Ampicillin, bacitracin carbapenems cephalosporin
• Methicillin, oxacillin, vancomycin
Rod-shaped bacterium before
penicillin.
The bacterial cell lysing as penicillin
weakens the cell wall.
The action of antimicrobial drugs
2. Injury to plasma membrane
– alter permeability
– toxicity risk - all membranes similarity
– Example: polymixin B
The action of antimicrobial drugs
3. Inhibition of proteins synthesis
– eukaryotes = 80s ribosome, prokaryotes = 70s
– can target 70s but toxicity to mitochondria
– inhibit peptide bond formation, attachment of tRNA, or cause misreading of
mRNA
– broad spectrum
– Examples: streptomycin, tetracycline, chloramphenicol
The action of antimicrobial drugs
4. Inhibition of nucleic acid synthesis
– Interfere with DNA replication or transcription (RNA synthesis)
– Examples: the quinolines, rifampin
5. Competitive inhibitors of the synthesis of essential metabolites
– competitive inhibitor of enzyme in metabolite synthesis pathway
– broad spectrum
– Examples: sulfonamides
Figure 20.13 - Overview
Antimicrobial drugs activity
• Disk-Diffusion Test (Kirby-Bauer test) is used in determining what
treatment of antibiotics should be used when treating an infection
– to determine the inhibition of bacterial growth measured under
standard conditions
– method:
• a culture medium - the Mueller-Hinton agar
• plate lawn of bacteria
• place disks of known antimicrobials on lawn
• measure zones of inhibition - The measurement
is compared to the criteria set by the National
Committee for Clinical Laboratory Studies (NCCLS).
• compare to a standard table for that drug
– Sensitive
– Intermediate
– Resistant
Figure 20.17
Antimicrobial drugs activity
• E Test
– to estimate MIC (minimal inhibitory concentration) for effective dose
Figure 20.18
Antimicrobial drugs activity
• Broth Dilution Test
– to estimate MIC (minimal inhibitory concentration)
– to estimate MBC (Minimal bactericidal concentration)
Figure 20.19
Antibiotic Resistance
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General mechanisms by which a microorganism may express
resistance to an antibiotic
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All are consequences of microbial gene expression
A variety of mutations can lead to antibiotic resistance.
1. Inactivation of the drug (Enzymatic destruction of the drug)
2. Prevention of penetration of the drug to the target site within
the microbe (changes in plasma or other membrane
permeability or formation of capsules)
3. Alteration of drug's target site (such as ribosomal proteins)
4. Rapid rejection of the drug
Antibiotic Resistance
• Resistance genes are often on plasmids or
transposons that can be transferred between bacteria.
• Improper use of antibiotics selects for resistance mutants.
• Improper use includes:
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Using outdated, weakened antibiotics
Using antibiotics for the common cold and other inappropriate conditions
Use of antibiotics in animal feed
Failure to complete the prescribed regimen
Using someone else's leftover prescription
Antibiotic Resistance
Figure 20.20
Effects of Combinations of Drugs
• Synergism occurs when the effect of two drugs together is
greater than the effect of either alone.
• Antagonism occurs when the effect of two drugs together is
less than the effect of either alone.
The Future of Chemotherapeutic Agents
• Antimicrobial peptides
– Broad spectrum antibiotics from plants and animals
• Squalamine (sharks)
• Protegrin (pigs)
• Magainin (frogs)
• Antisense agents
– Complementary DNA short nucleic acids
that binds to a pathogen's virulence gene(s)
and prevents transcription
• siRNA
– Complementary RNA that binds mRNA
to inhibit translation
Learning objectives
• Name the microbes that produce most antibiotics.
• Describe the problems of chemotherapy for viral, fungal,
protozoan, and helminthic infections.
• Define the following terms: spectrum of activity, broad-spectrum
drugs, superinfection.
• Identify five modes of action of antimicrobial drugs.
• List the advantages of each of the following over penicillin:
semisynthetic penicillins, cephalosporins, and vancomycin.
• Describe how each of the following inhibits protein synthesis:
aminoglycosides, tetracyclines, chloramphenicol, macrolides.
• Describe how rifamycins and quinolones kill bacteria.
• Describe how sulfa drugs inhibit microbial growth.
• Describe two tests for microbial susceptibility to chemotherapeutic
agents.
• Describe the mechanisms of drug resistance