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Sterilization, Disinfection
and Antibacterial Agents
• Pin Lin (凌 斌), Ph.D.
Departg ment of Microbiology & Immunology, NCKU
ext 5632
[email protected]
• References:
1. Chapters 10 & 20 in Medical Microbiology (Murray,
P. R. et al; 5th edition)
2. 醫用微生物學 (王聖予 等編譯, 4th edition)
Outline
1. Sterilization (Definition & Methods)
2. Disinfection (Definition & Methods)
3. Mechanisms of Antimicrobial Action
4. Antibacterial Agents
What is Sterilization?
Sterilization (in Microbiology) :
1. To completely remove all kinds of
microbes (bacteria, mycobacteria,
viruses, & fungi) by physical or chemical
methods.
2. Effective to kill “bacterium spores”
3. Sterilant: material or method used to
remove or kill all microbes
Methods of sterilization (I)
Methods of sterilization (II)
Sterilize instruments
Physical methods
(moist heat, dry heat, filtration, radiation)
Moist heat
Boiling: boiling for 10 min => Kill most vegetative forms of bacteria
Longer time => Kill spores
Addition of 2% Na2CO3 or 0.1% NaOH => enhance
destruction of spores and prevent rusting of the metal wares.
Low temperature disinfection (Pasteurization): 62-65 oC for 30 min.
or 71.5 oC for 15 sec. This is mainly used for disinfection of milk.
Autoclave: 121-132 oC for 15 min or longer => Kill both the vegetative
and spore forms of the bacteria.
=> Use Bacillius stearothermophilus spores to monitor the
effectiveness of Autoclave
Dry heat
Dry oven: 160 oC for 2 hrs or 171 oC for 1 hr. (B. subtilis)
Flaming; incineration
Radiation
UV-light: UV-radiation causes
damage to DNA.
Ionizing radiation: less applicable.
Filtration
The pore size for filtering bacteria,
yeasts, and fungi is in the range of
0.22-0.45 mm (filtration membranes
are most popular for this purpose).
HEPA filters
Chemical methods
Alcohol: protein denaturant. 70% aqueous solution of
ethyl alcohol and isopropyl alcohol are commonly used
as skin disinfectants.
Phenolics: Phenol and phenolic compounds (e.g. lysol)
lyse the cell membrane and denature proteins at 1-2%
(aqueous solution).
Oxidizing agents: inactivate cells by oxidizing free
sulfhydryl group, e.g., peracetic acid, iodine, iodophore,
H2O2 (3-6%), hypochlorite, and chlorine etc.
Plasma gas sterilization: H2O2 vapors treated with
microwave or radio energy to produce reactive free
radicals; no toxic byproducts. An efficient sterilization for
dry surfaces.
Alkylating agents
Formalin (37% aqueous solution of formaldehyde), glutaraldehyde
Ethylene oxide gas (made nonexplosive by mixing with CO2 or a
fluorocarbon): a reliable disinfectant for dry surfaces.
Detergents: surface-active agents that
disrupt the cell membranes.
Anionic detergents: e.g. soaps, and bile
salts.
Cationic detergents: e.g., the quaternary
ammonium compounds, are highly
bactericidal for both the gram-positive
and negative bacteria in the absence
of contaminating organic matter.
Pros & Cons of Sterilants (I)
1. Steam (Moist) & Dry Heat => the most common methods
for most materials.
Cons: NO good for heat-senstive, toxic or volatile
chemicals
2. Filtration => remove bacteria and fungi from air or
solutions
eg.: HEPA (High-Efficiency Particular Air) filters
Cons: unable to remove viruses and some small bacteria
(microplasma)
3. Ethylene oxide => the most common gas vapor sterilant
Cons: (1) flammable & explosive (2) potential carcinogenic
4. Formaldehyde gas => carcinogenic
Pros & Cons of Sterilants (II)
5. Plasma gas => Hydrogen peroxide => Reactive free
radicals
Microwave - or
=> No Toxic byproducts
radio-freq energy
=> may replace many applications for ethylene oxide
Cons: NOT good for materials absorbing or reacting
with H2O2
6. Peracetic acid => an oxidizing agent w/ good activity
=> end products nontoxic
7. Glutaraldehyde => Not safe
What is Disinfection?
Disinfection (in Microbiology) :
1. To kill most of microbial forms except some
resistant organisms or bacterium spores
2. Categorizing: High-level  sterilization
Intermediate-level
Low level
Not effective for
all bacteria
or spores
3. Disinfectant: a substance or method used to kill
microbes on surfaces
High-level disinfectants
Used for items involved in invasive procedures but NOT
withstand sterilization, e.g. Endoscopes, Surgical instruments
Intermediate-level disinfectants
Used for cleaning surface or instruments without bacterial
spores and highly resilient organism, eg. Laryngoscopes,
Anesthesia breathing circuits…etc
Low-level disinfectants
Used to treat non-critical instruments and devices, not
penetrating into mucosa surfaces or sterile tissues
Considerations of Disinfection
Effectiveness of disinfectants is influenced by:
1. Nature of the item to be disinfected
2. Number and resilience of the contaminants
3. Amount of organic material present
4. Type and concentration of disinfectant
5. Duration and temperature of exposure
Antisepsis & Antiseptic agents
1. Use of chemical agents on skin or living tissues to inhibit
or eliminate microbes
2. Antiseptic agents are selected for their safety & efficacy
Outline
• Introduction
• Sterilization (Definition & Methods)
• Disinfection (Definition & Methods)
• Mechanisms of Antimicrobial Action
The Discovery of Antibacterial
Agents
1.
In 1930s Gerhard Domagk discovered the antibacterial effect of prontosil (=> sulfanilamide) => 1939
Nobel Laureate
2. A. Fleming discovered that the mold Penicillium
prevented the multiplication of staphyloocci.
=> The first antibiotic, Penicillin, was identified => 1945
Nobel Laureate
3. Streptomycin, tetracyclines & others were thereafter
developed to treat infectious diseases.
4. Bacteria start developing resistance to these agents.
Mechanisms of Antibacterial agents
1. A useful chemotherapeutic agent should have in vivo
effectiveness and selective toxicity.
2. Modes of action of the chemotherapeutic agents
Inhibition of:
- Cell wall synthesis
- Protein synthesis
- Nucleic acid synthesis
- Cell membrane function
Sites of Action of Antibacterial
Chemical Agents
Peptidoglycan
1. A major component of cell wall
2. Forms a meshlike layer
consisting:
a polysaccharide polymer
cross-linked by Peptide bonds
3. Cross-linking reaction is
mediated by:
Transpeptidases
DD-carboxypeptidases
=> Targets of Penicillin
Outer wall of Gram-positive and
Gram-negative species
Inhibition of cell wall synthesis
(Penicillins, Cephalosporins, Vancomycin, Cycloserine, Bacitracin)
b-lactam drugs:
Drugs containing a b-lactam
ring, e.g. penicillins and
cephalosporins.
Vancomycin: bactericidal for
some gram-positive bacteria
PBPs (penicillin-binding proteins):
receptors for b-lactam drugs. There
are 3-6 PBPs, some of which are
transpeptidation enzymes.
Outline
• Introduction
• Sterilization (Definition & Methods)
• Disinfection (Definition & Methods)
• Mechanisms of Antimicrobial Action
• Antibacterial Agents
Penicillins
Produced by Penicillium chrysogenum
Modifications:
decrease acid lability;
increase absorption;
resistant to penicillinase;
broader spectrum (e.g., Ampicillin).
b-lactamase inhibitors: bind b-lactamases
irreversibly; combined use with some penicillins to
increase effectiveness.
Modifications of cephalosporins were to expand their
spectra or increase their stability to b-lactamases.
Vancomycin
1. A complex glycopeptide produced by Streptomyces
orientalis
2. Interacts with D-ala-D-ala termini of the pentapeptide
side chains
3. Inactive for gram-negative bacteria
4. Some enterococci have acquired resistance to
vancomycin
5. The resistance genes are carried on plasmids.
Polymyxins
1. Cyclic polypeptides (from Bacillus polymyxa)
2. Insert into bacteria outer membrane by interacting with
LPS and phospholipids  increase cell permeability
 bacterial cell death
3. Most Active for gram-negative bacteria, because Grampos bacteria have no outer member
4. Nephrotoxic
5. External treatment of localized infection and oral
administration to sterilize the gut.
Drug resistance of the microbes
1. Producing enzymes that degrade or modify the
active drugs;
2. Decreasing drug entry;
3. Increasing drug efflux;
4. Increasing the amount of target enzyme;
5. Decreasing affinity of target for drug;
6. Developing an altered metabolic pathway that
bypass the reaction inhibited by the drug.
How Bacteria Become Resistant to the
b-Lactam Antibiotics?
1. To prevent the interaction between the antibiotic and the target PBP
e.g. Gram-neg (Pseudomonas) => change porins on the pores =>
exclude antibiotic
2. To modify the binding of the antibiotic to the target PBP
Modified PBP can result from mutation or acquisition of new
PBP
3. Hydrolysis of the antibiotic by b-lactamases
- They are in the same family of PBPs
- Over 200 different b-lactamases:
some are specific for penicillins
others have a broad range of activity
Inhibition of protein synthesis
Aminoglycosides (streptomycin, kanamycin, neomycin, gentamicin,
tobramycin, amikacin, etc.): bind irreversibly to 30S ribosomal proteins
and inhibit peptide formation; bactericidal. Gm and Tm are ototoxic.
Tetracyclines: inhibit attachment of charged tRNA; bacteriostatic.
Chloramphenicol: binds to peptidyl transferase of ribosome; toxic to
bone marrow cells (aplastic anemia); bacteriostatic.
Macrolides (erythromycins, clarithromycin, etc.): bind to 23S rRNA and
block peptide elongation.
Lincomycins (clindamycin): resembles macrolides in mode of action.
Oxazolidinones (linezolid): blocks formation of imitiation complex.
Active against staphylococci, streptococci and enterococci. No crossresistance with the above antibiotics. Reserved for multidrug-resistant
enterococci.
Resistance to aminoglycosides:
1. Mutation to ribosomal binding site;
2. Decreased uptake of antibiotic;
3. Enzymatic modification of the antibiotic.
Inhibition of nucleic acid synthesis
quinolones, rifampin, sulfonamides, trimethoprime, pyrimethamine
Rifampin: inhibits RNA synthesis.
Quinolones and fluoquinolones: blocking DNA gyrase.
Metronidazol: effective to anaerobic bacterial infections.
Reduction of its nitro group by bacterial nitroreductase
produces cytotoxic compound that disrupts bacterial DNA.
Antimetabolites
Sulfonamides: analogs of p-aminobenzoic acid (PABA) and
inhibit synthesis of folic acid, which is an important precursor
to the synthesis of nucleic acids.
Trimethoprim: inhibits dihydrofolic acid reductase in synthesis
of purines, methionine and glycine.
Antimicrobial activity in vivo
Factors affecting the effectiveness of antibiotics
in vivo
Environment
Amount of pathogen
State of bacterial
metabolic activity
Distribution of drug
Location of organisms
Interfering substances
Concentration of
antibiotic
Absorption
Distribution
Variability of
concentration
Dangers of indiscriminate use of antibiotics
1. Development of drug resistance.
2. “Superinfection" resulting from changes in the normal
flora of the body.
3. Masking serious infection without eradicating it.
4. Drug toxicity.
5. Widespread sensitization of the population with
resulting hypersensitivity, anaphylaxis, rashes, fever,
blood disorders, cholestatic hepatitis, and perhaps
collagen-vascular diseases.
Genetic origin of drug resistance
Chromosomal
Extrachromosomal (e.g.,
R plasmids)
Can be transferred by
conjugation,
transformation, and
transduction.
A general rule in antimicrobial therapy
give a sufficiently large amount of an effective drug as early
as possible and continue treatment long enough to ensure
eradication of infection, but give an antimicrobial drug only
when it is indicated by rational choice.
Limitation of drug resistance
1. Maintain sufficiently high levels of the drug in the tissue to
inhibit both the original population and first-step mutants.
2. Simultaneously administer two drugs that do not give
cross-resistance.
3. Avoid exposure of microbes to a particular drug by limiting
its use, especially in hospitals and in animal feeds.
Cross-resistance: microbes resistant to a certain drug may
also be resistant to other drugs that share a mechanism of
action. (e.g., different aminoglycosides, macrolides, and
lincomycins)
Selection of antibiotics
Diagnosis
Antibiotic susceptibility tests
Antimicrobial drugs used in combination
Indications
Prompt treatment of patients suspected of having a serious
microbial infection.
To delay the emergence of mutants resistant to one drug in chronic
infections.
To treat mixed infections.
To achieve bactericidal synergism or to provide bactericidal action.
Disadvantages
Relaxation of the effort to establish a diagnosis.
Greater chance for adverse reactions.
Unnecessary cost.
Not necessarily effective than single drug treatment.
Antagonism between drugs (rarely).
Effects of combined usage of two antibiotics
Indifference
(A + B=A or B)
Addition
(A + B=A + B)
Synergism
(A + B=A x B)
Antagonism
(A + B= 0 or less)
SUMMARY-I
1. Various antimicrobial agents act by interfering with:
(1) cell wall synthesis, (2) plasma membrane integrity,
(3) nucleic acid synthesis, (4) ribosomal function,
and (5) metabolite synthesis.
2. Cell wall synthesis is inhibited by ß-lactams, such as
penicillins and cephalosporins, which inhibit
peptidoglycan polymerization, and by vancomycin, which
combines with cell wall substrates.
SUMMARY-II
3. Bacteria can evolve resistance to antibiotics.
Resistance factors can be encoded on plasmids or on the
chromosome. Resistance may (1) decreased entry of the
drug, (2) changes in the receptor (target) of the drug,
or (3) metabolic inactivation of the drug.
4. Combinations of antibiotics may act synergisticallyproducing an effect stronger than the sum of the
effects of the two drugs alone or antagonistically, if one
agent inhibits the effect of the other.
Disinfection and Sterilization
Disinfection: killing of most microbial forms.
Disinfectant: a chemical substance used to kill microbes on surfaces
but too toxic to be applied directly to tissue.
Antisepsis: inhibit or eliminate microbes on skin or other living tissue
Sterilization: removal of life of every kind by physical or chemical
methods.
Sterilant: an agent or method used to remove or kill all microbes.
Septic: presence of pathogenic microbes in living tissue.
Aseptic: absence of pathogenic microbes.
Sterile: free of life of every kind.
Bacteriostatic: inhibiting bacterial multiplication. Bacteriostatic action
is reversible by removal or inactivation of agent.
Bactericidal: killing bacteria.
Modes of action of antimicrobial agents
1. Damage to DNA
Formation of pyrimidine dimer by UV irradiation
Single- or double-strand DNA break by ionizing radiation
DNA reactive chemicals, e.g. alkylating agents
2. Protein denaturation
3. Disruption of cell membrane or wall
4. Removal of free sulfhydryl groups
Formation of disulfide bond by oxidizing agents
Heavy metals combine with sulfhydryls
5. Chemical antagonism: interference with the normal
reaction between an enzyme and its substrate.
Disruption
of cell wall
Sites of antibiotic
activity