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 (5 th edition)
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Department of Microbiology & Immunology, NCKU
1. Murray, P. et al., Medical Microbiology, Ch10 (5th edition)
2. Samuel Baron, Medical Microbiology Ch11 (4th edition)
Methods of sterilization (I)
Methods of sterilization (II)
Microwave - or radio-freq energy
Not effective for
Used for items involved in invasive procedures but NOT withstand sterilization, e.g. Endoscopes, Surgical instruments
Used for cleaning surface or instruments without bacterial spores and highly resilient organism, eg. Laryngoscopes, Anesthesia breathing circuits…etc
Used to treat noncritical instruments and devices, not penetrating into mucosa surfaces or sterile tissues
Considerations of Disinfection
Antisepsis & Antiseptic agents
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 oven: 160 oC for 2 hrs or 171 oC for 1 hr. (B. subtilis)
UV-light: UV-radiation causes damage to DNA.
Ionizing radiation: less applicable.
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).
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.
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.
The Discovery of Antibacterial Agents
=> The first antibiotic, Penicillin, was identified => 1945 Nobel Laureate
1. A useful chemotherapeutic agent should havein vivo effectivenessand selective toxicity.
2. Modes of action of the chemotherapeutic agents
cell wall synthesis
nucleic acid synthesis
(cell membrane function)
Sites of Action of Antibacterial Chemical Agents
Outer wall of Gram-positive and Gram-negative species
Inhibition of cell wall synthesis(penicillins, cephalosporins, vancomycin, cycloserine, bacitracin)
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.
Produced by Penicillium chrysogenum
decrease acid lability;
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.
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
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
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?
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 cross-resistance with the above antibiotics. Reserved for multidrug-resistant enterococci.
Inhibition of nucleic acid synthesisquinolones, 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.
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
Concentration of antibiotic
Variability of concentration
Amount of pathogen
State of bacterial metabolic activity
Distribution of drug
Location of organisms
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
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
Antibiotic susceptibility tests
Antimicrobial drugs used in combination
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.
Relaxation of the effort to establish a diagnosis.
Greater chance for adverse reactions.
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)
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
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.
In persons of normal susceptibility exposed to a specific pathogen
In persons of increased susceptibility
B. stearothermophilus spores
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: 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.
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.
: N-acetylmuraminic acid