Sterilization disinfection and antibacterial agents
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Sterilization, Disinfection and Antibacterial Agents. 王淑鶯 微生物免疫學所 國立成功大學醫學院 分機 : 5634 Email: [email protected] Outline. Definition of Sterilization and Disinfection Physical and Chemical Methods of Antimicrobial Control Antibiotics and Mechanisms of Antimicrobial Action

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Sterilization disinfection and antibacterial agents

Sterilization, Disinfection and Antibacterial Agents

王淑鶯

微生物免疫學所

國立成功大學醫學院

分機: 5634

Email: [email protected]


Outline

Outline

  • Definition of Sterilization and Disinfection

  • Physical and Chemical Methods of Antimicrobial Control

  • Antibiotics and Mechanisms of Antimicrobial Action

    Reference:

    Chapters 8 & 20 in Medical Microbiology

    (Murray, P. R. et al; 6th edition)


Sterilization disinfection and antibacterial agents

Joseph Lister (5 April 1827 – 10 February 1912) was an English surgeon and a pioneer of antiseptic surgery, who promoted the idea of sterile surgery. Lister successfully introduced carbolic acid to sterilize surgical instruments and to clean wounds, which led to reduced post-operative infections and made surgery safer for patients.

http://en.wikipedia.org/wiki/Joseph_Lister,_1st_Baron_Lister


Sterilization disinfection and antibacterial agents

While he was a professor of surgery at the University of Glasgow, Lister became aware of a paper published by the French chemist Louis Pasteur showing that rotting and fermentation could occur under anaerobic conditions if micro-organisms were present. Pasteur suggested three methods to eliminate the microorganisms responsible for gangrene: filtration, exposure to heat, or exposure to chemical solutions. Lister confirmed Pasteur's conclusions with his own experiments and decided to use his findings to develop antiseptic techniques for wounds. As the first two methods suggested by Pasteur were inappropriate for the treatment of human tissue, Lister experimented with the third.

http://en.wikipedia.org/wiki/Joseph_Lister,_1st_Baron_Lister


Sterilization disinfection and antibacterial agents

Carbolic acid (phenol) had been in use as a means of deodorising sewage, so Lister tested the results of spraying instruments, the surgical incisions, and dressings with a solution of it. Lister found that carbolic acid solution swabbed on wounds remarkably reduced the incidence of gangrene.

http://en.wikipedia.org/wiki/Joseph_Lister,_1st_Baron_Lister


Sterilization disinfection and antibacterial agents

Lister also noticed that midwife-delivered babies had a lower mortality rate than surgeon-delivered babies, correctly attributing this difference to the fact that midwives tended to wash their hands more often than surgeons, and that surgeons often would go directly from one surgery, such as draining an abscess, to delivering a baby. He instructed surgeons under his responsibility to wear clean gloves and wash their hands before and after operations with 5% carbolic acid solutions.

http://en.wikipedia.org/wiki/Joseph_Lister,_1st_Baron_Lister


Sterilization disinfection and antibacterial agents

Before then:

  • Nosocomial infections caused death in 10% of surgeries.

  • Up to 25% mothers delivering in hospitals died due to infection.


Antimicrobial definitions

Antimicrobial Definitions

  • Sterilization

    • To completely remove all kinds of microbes (bacteria, mycobacteria, viruses, & fungi) by physical or chemical methods

    • Effective to kill “bacterial spores”

    • Sterilant: material or method used to remove or kill all microbes


Antimicrobial definitions1

Antimicrobial Definitions

  • Disinfection

    • To reduce the number of pathogenic microorganisms to the point where they no longer cause diseases

    • Usually involves the removal of vegetative or non-endosporeforming pathogens

    • May use physical or chemical methods

      • Disinfectant: An agent applied to inanimate objects.

      • Antiseptic: A substance applied to living tissue.

      • Sanitization: Use of chemical agent on food-handling equipment to meet public health standards and minimize chances of disease transmission. E.g: Hot soap & water


Antimicrobial definitions2

Antimicrobial Definitions

  • Bacteriostatic

    • prevents growth of bacteria

  • Germicide

    • An agent that kills certain microorganisms.

      • Bactericide: An agent that kills bacteria. Most do not kill endospores.

      • Viricide: An agent that inactivates viruses.

      • Fungicide: An agent that kills fungi.


Method of control

Method of Control

  • physical or chemical?

    • physical control includes heat, irradiation, filtration and mechanical removal

    • chemical control involves the use of antimicrobial chemicals

    • depends on the situation

    • degree of control required

antimicrobial chemicals

air filters


Factors influence the effectiveness of antimicrobial treatment

Factors influence the effectiveness of antimicrobial treatment

  • Number of Microbes: The more microbes present, the more time it takes to eliminate population.

  • Type of Microbes: Endospores are very difficult to destroy. Vegetative pathogens vary widely in susceptibility to different methods of microbial control.

  • Environmental influences: Presence of organic material (blood, feces, saliva, pH etc.) tends to inhibit antimicrobials.

  • Time of Exposure: Chemical antimicrobials and radiation treatments are more effective at longer times. In heat treatments, longer exposure compensates for lower temperatures.


Rate of microbial death

Rate of Microbial Death

When bacterial populations are heated or treated antimicrobial chemicals, they usually die at a constant rate.


Physical methods of microbial control

Physical Methods of Microbial Control

  • heat

  • filtration

  • radiation


Physical methods of microbial control1

Physical Methods of Microbial Control

  • Heat

    • Kills microorganisms by denaturing their enzymes and other proteins. Heat resistance varies widely among microbes.

    • fast, reliable, inexpensive

    • does not introduce potential toxic substances

  • types of heat control include

    • moist heat

    • pasteurization

    • dry heat


Physical methods of microbial control2

Physical Methods of Microbial Control

Moist Heat: Kills microorganisms by coagulating their proteins.

  • Boiling: Heat to 100oC or more at sea level. Kills vegetative forms of bacterial pathogens. Most pathogens can be killed within 10 minutes or less. Endospores and some viruses are not destroyed this quickly.

  • In general, moist heat is much more effective than dry heat.


Physical methods of microbial control3

Physical Methods of Microbial Control

Moist Heat (Continued):

Reliable sterilization with moist heat requires temperatures above that of boiling water.

  • Autoclave: Chamber which is filled with hot steam under pressure. Preferred method of sterilization, unless material is damaged by heat, moisture, or high pressure.

    • Temperature of steam reaches 121oC at twice atmospheric pressure.

    • All organisms and endospores are killed within 15 minutes.


Autoclave closed chamber with high temperature and pressure

Autoclave: Closed Chamber with High Temperature and Pressure


Physical methods of microbial control4

Physical Methods of Microbial Control

Moist Heat (Continued):

  • Pasteurization: Developed by Louis Pasteur to prevent the spoilage of beverages. Used to reduce microbes responsible for spoilage of beer, milk, wine, juices, etc.

    • Classic Method of Pasteurization: Milk was exposed to 65oC for 30 minutes.

    • High Temperature Short Time Pasteurization (HTST): Used today. Milk is exposed to 72oC for 15 seconds.


Physical methods of microbial control5

Physical Methods of Microbial Control

Dry Heat:

  • Direct Flaming: Used to sterilize inoculating loops and needles. Heat metal until it has a red glow.

  • Incineration: Effective way to sterilize disposable items (paper cups, dressings) and biological waste.

  • Hot Air Sterilization: Place objects in an oven. Require 2 hours at 170oC for sterilization. Dry heat is transfers heat less effectively to a cool body, than moist heat.


Physical methods of microbial control6

Physical Methods of Microbial Control

Filtration: Removal of microbes by passage of a liquid or gas through a screen like material with small pores. Used to sterilize heat sensitive materials like vaccines, enzymes, antibiotics, and some culture media.

  • Membrane Filters: Uniform pore size. Used in industry and research. Different sizes:

    • 0.22 and 0.45um Pores: Used to filter most bacteria. Don’t retain spirochetes, mycoplasmas and viruses.

    • 0.01 um Pores: Retain all viruses and some large proteins.

  • High Efficiency Particulate Air Filters (HEPA): Used in operating rooms to remove bacteria from air.


Physical methods of microbial control7

Physical Methods of Microbial Control

Filtration

  • used for heat sensitive fluids

  • air


Physical methods of microbial control8

Physical Methods of Microbial Control

Low Temperature: Effect depends on microbe and treatment applied.

  • Refrigeration: Temperatures from 0 to 7oC. Bacteriostaticeffect. Reduces metabolic rate of most microbes so they cannot reproduce or produce toxins.

  • Freezing: Temperatures below 0oC.


Physical methods of microbial control9

Physical Methods of Microbial Control

Desiccation: In the absence of water, microbes cannot grow or reproduce, but some may remain viable for years. After water becomes available, they start growing again.

Susceptibility to desiccation varies widely:

  • Neisseria gonnorrhea: Only survives about one hour.

  • Mycobacterium tuberculosis: May survive several months.

  • Viruses are fairly resistant to desiccation.

  • Clostridium spp. and Bacillus spp.: May survive decades.


Physical methods of microbial control10

Physical Methods of Microbial Control

Osmotic Pressure: The use of high concentrations of salts and sugars in foods is used to increase the osmotic pressure and create a hypertonic environment.

Plasmolysis: As water leaves the cell, plasma membrane shrinks away from cell wall.

  • Yeasts and molds: More resistant to high osmotic pressures.

  • Staphylococci spp. that live on skin are fairly resistant to high osmotic pressure.


Physical methods of microbial control11

Physical Methods of Microbial Control

Radiation: Three types of radiation kill microbes:

1. Ionizing Radiation: Gamma rays, X rays, electron beams, or higher energy rays. Have short wavelengths (less than 1 nanometer).

Used to sterilize pharmaceuticals, disposable medical supplies and food.

Disadvantages: Penetrates human tissues. May cause genetic mutations in humans.


Forms of radiation

Forms of Radiation


Physical methods of microbial control12

Physical Methods of Microbial Control

Radiation: Three types of radiation kill microbes:

2. Ultraviolet light (Nonionizing Radiation): Wavelength is longer than 1 nanometer. Damages DNA by producing thymine dimers, which cause mutations.

Used to disinfect operating rooms, nurseries, cafeterias.

Disadvantages: Damages skin, eyes. Doesn’t penetrate paper, glass, and cloth.


Physical methods of microbial control13

Physical Methods of Microbial Control

Radiation: Three types of radiation kill microbes:

3.Microwave Radiation: Wavelength ranges from 1 millimeter to 1 meter.

Heat is absorbed by water molecules.

May kill vegetative cells in moist foods.

Bacterial endospores, which do not contain water, are not damaged by microwave radiation.

Solid foods are unevenly penetrated by microwaves.


Chemical methods of microbial control types of disinfectants

Chemical Methods of Microbial ControlTypes of Disinfectants

1. Phenols and Phenolics:

  • Phenol (carbolic acid) was first used by Lister as a disinfectant.

    • Rarely used today because it is a skin irritant and has strong odor.

  • Phenolics are chemical derivatives of phenol

    • Cresols (Lysol): Derived from coal tar.

    • Bisphenols: Effective against gram-positive staphylococci and streptococci. Excessive use in infants may cause neurological damage. E.g. hexachlorophene

  • Destroy plasma membranes and denature proteins.

  • Advantages: Stable, persist for long times after applied, and remain active in the presence of organic compounds.


Chemical methods of microbial control types of disinfectants1

Chemical Methods of Microbial ControlTypes of Disinfectants

2. Halogens: Effective alone or in compounds.

A.Iodine:

  • Iodine tincture (alcohol solution) was one of first antiseptics used.

  • Precipitates proteins and oxidizes essential enzymes

    B. Chlorine:

  • When mixed in water forms hypochlorousacid:

    Cl2 + H2O ------>H+ + Cl-+ HOCl

    Hypochlorousacid

  • Used to disinfect drinking water, pools, and sewage.


Chemical methods of microbial control types of disinfectants2

Chemical Methods of Microbial ControlTypes of Disinfectants

3. Alcohols:

  • Kill bacteria, fungi, but not endospores or viruses.

  • Act by denaturing proteins and disrupting cell membranes.

  • Used to mechanically wipe microbes off skin before injections or blood drawing.

  • Not good for open wounds, because cause proteins to coagulate.

    • Ethanol: Drinking alcohol. Optimum concentration is 70%.

    • Isopropanol: Rubbing alcohol. Better disinfectant than ethanol. Also cheaper and less volatile.


Chemical methods of microbial control types of disinfectants3

Chemical Methods of Microbial ControlTypes of Disinfectants

4. Quaternary Ammonium Compounds (Quats):

  • Cationic (positively charge) compound acts like detergents.

  • Denatures cell membranes

  • Effective against gram positive bacteria, less effective against gram-negative bacteria.

  • Benzalkonium chloride, cetylpyridinium chloride


Chemical methods of microbial control types of disinfectants4

Chemical Methods of Microbial ControlTypes of Disinfectants

5. Aldehydes:

  • Include some of the most effective antimicrobials.

  • Inactivate proteins by forming covalent crosslinks with several functional groups.

    A. Formaldehyde:

  • Excellent disinfectant, 2% aqueous solution.

  • Commonly used as formalin, a 37% aqueous solution.

  • Formalin was used extensively to preserve biological specimens and inactivate viruses and bacteria in vaccines.

  • Irritates mucous membranes, strong odor.


Chemical methods of microbial control types of disinfectants5

Chemical Methods of Microbial ControlTypes of Disinfectants

5. Aldehydes:

B. Glutaraldehyde:

  • Less irritating and more effective than formaldehyde.

    6. Gaseous Sterilizers:

  • Chemicals that sterilize in a chamber similar to an autoclave.

  • Denature proteins, by replacing functional groups with alkyl groups.

    Ethylene Oxide:

  • Kills all microbes and endospores, but requires exposure of 4 to 18 hours.

  • Commonly used to disinfect hospital instruments


Chemical methods of microbial control types of disinfectants6

Chemical Methods of Microbial ControlTypes of Disinfectants

7. Oxidizing Agents:

  • Oxidize cellular components of treated microbes.

  • Disrupt membranes and proteins.

    A. Ozone:

  • Used along with chlorine to disinfect water.

  • Helps neutralize unpleasant tastes and odors.

  • More effective killing agent than chlorine, but less stable and more expensive.

  • Highly reactive form of oxygen.

  • Made by exposing oxygen to electricity or UV light

    B. Hydrogen Peroxide:

  • Not good for open wounds because quickly broken down by catalase present in human cells.

  • Effective in disinfection of inanimate objects


Outline1

Outline

  • Definition of Sterilization and Disinfection

  • Physical and Chemical Methods of Antimicrobial Control

  • Antibiotics and Mechanisms of Antimicrobial Action


Definition of an antibiotic

Definition of an Antibiotic

  • Substance produced by a microorganism or a similar product produced wholly (synthetic) or partially (semi-synthetic) by chemical synthesis and in low concentrations inhibits the growth of or kills microorganisms.


Microbial sources of antibiotics

Microbial Sources of Antibiotics


Antibiotic spectrum of activity

Antibiotic Spectrum of Activity

  • No antibiotic is effective against all microbes


Mechanisms of antimicrobial action

Mechanisms of Antimicrobial Action

  • Bacteria have their own enzymes for

    • Cell wall formation

    • Protein synthesis

    • DNA replication

    • RNA synthesis

    • Synthesis of essential metabolites


Modes of antimicrobial action

Modes of Antimicrobial Action


Antibacterial antibiotics inhibitors of cell wall synthesis

Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis

  • Bacteria cell wall contains peptidoglycan

  • Antimicrobials that interfere with the synthesis of cell wall do not interfere with eukaryotic cell

  • Antimicrobials of this class include

    • β- lactam drugs

    • Vancomycin

    • Bacitracin


Antibacterial antibiotics inhibitors of cell wall synthesis1

Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis

  • Penicillins and Cephalosporins

    • Part of group of drugs called β –lactams

      • Have shared chemical structure called β-lactam ring

    • Competitively inhibits function of penicillin-binding proteins (involved in the final stages of the synthesis of peptidoglycan)

      • Inhibits peptide bridge formation between glycan molecules

      • This causes the cell wall to develop weak points at the growth sites and become fragile.


Antibacterial antibiotics inhibitors of cell wall synthesis2

Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis

  • The weakness in the cell wall causes the cell to lyze.


Antibacterial antibiotics inhibitors of cell wall synthesis3

Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis

  • Natural penicillins

    • Narrow range of action

    • Susceptible to penicillinase (b- lactamase)

  • Semisynthetic Penicillins

    • Penicilinase-resistant penicillins

      • Carbapenems: very broad spectrum

      • Monobactam: Gram negative

    • Extended-spectrum penicillins


  • Antibacterial antibiotics inhibitors of cell wall synthesis4

    Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis

    • Cephalosporins

      • chemical structures make them resistant to inactivation by certain β-lactamases

      • most effective against Gram – bacteria.

      • chemically modified to produce family of related compounds

        • 2nd, 3rd, and 4th generations more effective against gram-negatives


    Antibacterial antibiotics inhibitors of cell wall synthesis5

    Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis

    • Bacitracin

      • Interferes with transport of peptidoglycan precursors across cytoplasmic membrane

      • Toxicity limits use to topical applications

      • Common ingredient in non-prescription first-aid ointments


    Antibacterial antibiotics inhibitors of cell wall synthesis6

    Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis

    • Vancomycin

      • Inhibits formation of glycan chains

      • Important in treating infections caused by penicillin resistant Gram + organisms

      • Acquired resistance most often due to alterations in side chain of NAM molecule

        • Prevents binding of vancomycin to NAM component of glycan

      • Important "last line" against antibiotic resistant S. aureus


    Antibacterial antibiotics inhibitors of protein synthesis

    Antibacterial Antibiotics Inhibitors of Protein Synthesis

    • Inhibition of protein synthesis

      • Structure of prokaryotic ribosome acts as target for many antimicrobials of this class

        • Differences in prokaryotic and eukaryotic ribosomes responsible for selective toxicity

      • Drugs of this class include

        • Aminoglycosides

        • Tetracyclins

        • Macrolids

        • Chloramphenicol


    Antibacterial antibiotics inhibitors of protein synthesis1

    Antibacterial Antibiotics Inhibitors of Protein Synthesis

    • Aminoglycosides

      • binds to ribosomal subunits, causes misreading of mRNA

      • Examples of aminoglycosides include

        • Gentamicin, streptomycin and neomycin

      • Often used in synergistic combination with β-lactam drugs

        • Allows aminoglycosides to enter cells that are often resistant

      • Side effects

        • Nephrotoxicity


    Antibacterial antibiotics inhibitors of protein synthesis2

    Antibacterial Antibiotics Inhibitors of Protein Synthesis

    • Tetracyclins

      • Reversibly bind 30S ribosomal subunit

        • Blocks attachment of tRNA to ribosome

      • Effective against certain Gram + and Gram –

      • Can cause discoloration of teeth if taken as young child


    Antibacterial antibiotics inhibitors of protein synthesis3

    Antibacterial Antibiotics Inhibitors of Protein Synthesis

    • Macrolids

      • Reversibly binds to 50S ribosome

        • Prevents continuation of protein synthesis

      • Effective against variety of Gram + organisms and those responsible for atypical pneumonia

      • Often drug of choice for patients allergic to penicillin

      • Macrolids include

        • Erythromycin, clarithromycin and azithromycin


    Antibacterial antibiotics inhibitors of protein synthesis4

    Antibacterial Antibiotics Inhibitors of Protein Synthesis

    • Chloramphenicol

      • Binds to 50S ribosomal subunit

        • Prevents peptide bonds from forming and blocking proteins synthesis

      • Effective against a wide variety of organisms

      • Rare but lethal side effect is aplastic anemia


    Antibacterial antibiotics inhibitors of nucleic acid synthesis

    Antibacterial Antibiotics Inhibitors of Nucleic Acid Synthesis

    • Fluoroquinolones

      • Inhibit action of topoisomerase DNA gyrase

      • Examples include

        • Ciprofloxacin and ofloxacin

      • Urinary tract infections

    • Rifamycins

      • Block prokaryotic RNA polymerase

      • Primarily used to treat tuberculosis and preventing meningitis after exposure to N. meningitidis


    Antibacterial antibiotics inhibitors of metabolic pathway

    Antibacterial Antibiotics Inhibitors of Metabolic Pathway

    • Sulfonamides (sulfa drugs)

      • Inhibit folic acid synthesis

      • Structurally similar to para-aminobenzoic acid

        • Substrate in folic acid pathway

        • Through competitive inhibition of enzyme that aids in production of folic acid

      • Inhibit growth of Gram + and Gram - organisms


    Antibacterial antibiotics disruption of plasma membrane

    Antibacterial Antibiotics Disruption of Plasma Membrane

    • Polymyxin B

      • Binds membrane of Gram - cells

        • Alters permeability

          • Leads to leakage of cell and cell death

        • Also bind eukaryotic cells but to lesser extent

          • Limits use to topical application

      • Common ingredient in first-aid skin ointments


    Mechanisms of antibiotic resistance

    Mechanisms of Antibiotic Resistance

    • Enzymatic destruction of drug

      • Some organisms produce enzymes that chemically modify drug

        • Penicillinase breaks β-lactam ring of penicillin antibiotics

    • Alteration of drug's target site

      • Minor structural changes in antibiotic target can prevent binding

        • Changes in ribosomal RNA prevent macrolids from binding to ribosomal subunits


    Mechanisms of antibiotic resistance1

    Mechanisms of Antibiotic Resistance

    • Prevention of penetration of drug

      • Alterations in porin proteins decrease permeability of cells

        • Prevents certain drugs from entering

    • Rapid ejection of the drug

      • Some organisms produce efflux pumps

        • Increases overall capacity of organism to eliminate drug

          • Enables organism to resist higher concentrations of drug

          • Tetracycline resistance


    Effects of combinations of drugs

    EFFECTS OF COMBINATIONS OF DRUGS

    • Synergism

      • the chemotherapeutic effects of two drugs given simultaneously is greater than the effect of either given alone

      • For example, penicillin and streptomycin in the treatment of bacterial endocarditis. Damage to bacterial cell walls by penicillin makes it easier for streptomycin to enter


    Effects of combinations of drugs1

    EFFECTS OF COMBINATIONS OF DRUGS

    • Antagonism

      • the chemotherapeutic effects of two drugs given simultaneously reduce the effect of either given alone

      • For example, the simultaneous use of penicillin and tetracycline is often less effective than when wither drugs is used alone. By stopping the growth of the bacteria, the bacteriostatic drug tetracycline interferes with the action of penicillin, which requires bacterial growth.


    Summary

    Summary

    • Antimicrobial control

      • physical methods

      • chemical methods

    • Antibiotics

      • mechanisms of action


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