Download Physical and Chemical Methods of Disinfection and Sterilization

Sterilization, Disinfection and
Antibacterial Agents
• Pin Ling (凌 斌), Ph.D.
Department of Microbiology & Immunology, NCKU
ext 5632
[email protected]
• References:
1. Murray, P. et al., Medical Microbiology, Ch10 (5th edition)
2. Samuel Baron, Medical Microbiology Ch11 (4th edition)
Outline
• Sterilization (Definition & Methods)
• Disinfection (Definition & Methods)
• Mechanisms of Antimicrobial Action
• 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)
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)
1. Plasma gas => Hydrogen peroxide
Reactive free radicals
Microwave - or radio-freq energy
=> No Toxic byproducts
=> may replace many applications for ethylene oxide
Cons: NOT good for materials absorbing or reacting with
H 2 O2
2. Peracetic acid => an oxidizing agent w/ good activity
=> end products nontoxic
3. 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 noncritical 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
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.
Outline
• Introduction
• Sterilization (Definition & Methods)
• Disinfection (Definition & Methods)
• Mechanisms of Antimicrobial Action
• Antibacterial Agents
The Discovery of Antibacterial Agents
1.
In 1930s Gerhard Domagk discovered the anti-bacterial 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
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.
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
A complex glycopeptide produced by Streptomyces
orientalis
Interacts with D-ala-D-ala termini of the pentapeptide
side chains
Inactive for gram-negative bacteria
Some enterococci have acquired resistance to
vancomycin
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
Gram-pos 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
Mechanisms
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
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.
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 synergistically-producing 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.
Basis of Antimicrobial Action
Various antimicrobial agents act by interfering with (1) cell wall synthesis, (2) plasma membrane integrity, (3) nucleic acid
synthesis, (4) ribosomal function, and (5) folate synthesis.
Action of Specific Agents
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. Polymyxins disrupt the plasma membrane,
causing leakage. The plasma membrane sterols of fungi are attacked by polyenes (amphotericin) and imidazoles.
Quinolones bind to a bacterial complex of DNA and DNA gyrase, blocking DNA replication. Nitroimidazoles damage
DNA. Rifampin blocks RNA synthesis by binding to DNA directed RNA polymerase. Aminoglycosides, tetracycline,
chloramphenicol, erythromycin, and clindamycin all interfere with ribosome function. Sulfonamides and trimethoprim
block the synthesis of the folate needed for DNA replication
Bacterial Resistance
Bacteria can evolve resistance to antibiotics. Resistance factors can be encoded on plasmids or on the chromosome.
Resistance may involve decreased entry of the drug, changes in the receptor (target) of the drug, or metabolic
inactivation of the drug.
Effects of Combination Therapy
Combinations of antibiotics may act synergistically-producing 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.
Adverse Effects of Antimicrobial Agents
Many antibiotics are toxic to the host. Alterations of the normal intestinal flora caused by antibiotics may result in diarrhea
or in superinfection with opportunistic pathogens.
Antimicrobial chemoprophylaxis
In persons of normal susceptibility exposed
to a specific pathogen
In persons of increased susceptibility
In surgery
Back
B. stearothermophilus spores
Back
Aminoglycosides
Amino sugars
Aminocyclitol
Back
Back
Back
The earliest evidence of successful chemotherapy is from ancient Peru, where the Indians used
bark from the cinchona tree to treat malaria. Other substances were used in ancient China, and
we now know that many of the poultices used by primitive peoples contained antibacterial and
antifungal substances. Modern chemotherapy has been dated to the work of Paul Ehrlich in
Germany, who sought systematically to discover effective agents to treat trypanosomiasis and
syphilis. He discovered p-rosaniline, which has antitrypanosomal effects, and arsphenamine,
which is effective against syphilis. Ehrlich postulated that it would be possible to find
chemicals that were selectively toxic for parasites but not toxic to humans. This idea has been
called the "magic bullet" concept. It had little success until the 1930s, when Gerhard Domagk
discovered the protective effects of prontosil, the forerunner of sulfonamide. Ironically,
penicillin G was discovered fortuitously in 1929 by Fleming, who did not initially appreciate
the magnitude of his discovery. In 1939 Florey and colleagues at Oxford University again
isolated penicillin. In 1944 Waksman isolated streptomycin and subsequently found agents such
as chloramphenicol, tetracyclines, and erythromycin in soil samples. By the 1960s,
improvements in fermentation techniques and advances in medicinal chemistry permitted the
synthesis of many new chemotherapeutic agents by molecular modification of existing
compounds. Progress in the development of novel antibacterial agents has been great, but the
development of effective, nontoxic antifungal and antiviral agents has been slow. Amphotericin
B, isolated in the 1950s, remains an effective antifungal agent, although newer agents such as
fluconazole are now widely used. Nucleoside analogs such as acyclovir have proved effective in
the chemotherapy of selected viral infections.
Disruption
of cell wall
Sites of antibiotic
activity
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.
Peptidoglycan (of Staphylococcus aureus)
: N-acetylmuraminic acid
: -Ala-IGln-Lys-Ala-
: N-acetylglucosamine
: [Gly]5
Resistance to b-lactam antibiotics:
1. Prevention of interaction of drug and the target PBP;
2. Decrease binding of drug to PBP;
Modified PBP can result from mutation or acquisition of
new PBP
3. Hydrolysis of drug by producing b-lactamase (> 200
different kinds).