Sterilization, Disinfection and Antibacterial Agents

• Pin Lin ( 凌 斌 ), Ph.D.

Departg ment of Microbiology & Immunology, NCKU

ext 5632

lingpin@mail.ncku.edu.tw

• References:

1. Chapters 8 & 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 microb

es (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)

Not for virus

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

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

Dry heat

Dry oven: 160 oC for 2 hrs or 171 oC for 1 hr. (B. subtilis)

Chemical methods Alcohol: protein denaturant. 70% aqueous solution of e

thyl 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, H2

O2 (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 fluoro

carbon): 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.

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 solutionseg.: HEPA (High-Efficiency Particular Air) filtersCons: unable to remove viruses and some small bacteria (microplasma)

3. Ethylene oxide => the most common gas vapor sterilantCons: (1) flammable & explosive (2) potential carcinogenic

4. Formaldehyde gas => carcinogenic

Pros & Cons of Sterilants (I)

5. Plasma gas => Hydrogen peroxide => Reactive free radicals => No Toxic byproducts=> may replace many applications for ethylene oxideCons: 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

Pros & Cons of Sterilants (II)

Microwave - or radio-freq energy

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

3. Disinfectant: a substance or method used to kill microbes on surfaces

Not effective for all bacteria or spores

High-level disinfectants

Used for items involved in invasive procedures but NOT withstand sterilization, e.g. Endoscopes, Surgical instruments

Intermediate-level disinfectantsUsed for cleaning surface or instruments without bacterial spores and highly resilient organism, eg. Laryngoscopes, Anesthesia breathing circuits…etc

Low-level disinfectantsUsed 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 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.

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

Peptidoglycan1. 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

-lactam drugs:

Drugs containing a -lactam ring, e.g. penicillins and cephalosporins.

Vancomycin: bactericidal for some gram-positive bacteria

Inhibition of cell wall synthesis(Penicillins, Cephalosporins, Vancomycin, Cycloserine, Bacitrac

in)

PBPs (penicillin-binding proteins): receptors for -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

PenicillinsProduced by Penicillium chrysogenum

Modifications:

decrease acid lability;

increase absorption;

resistant to penicillinase;

broader spectrum (e.g., Ampicillin).

-lactamase inhibitors: bind -lactamases irreversibly; combined use with some penicillins to increase effectiveness.

Modifications of cephalosporins were to expand their spectra or increase their stability to -lactamases.

Vancomycin

1. A complex glycopeptide produced by Streptomyces or

ientalis

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 L

PS and phospholipids Increase cell permeabilit

y 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

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 -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 -lactamases - They are in the same family of PBPs - Over 200 different -lactamases:

some are specific for penicillinsothers have a broad range of activity

Outer wall of Gram-positive and Gram-negative species

Inhibition of protein synthesis Aminoglycosides (streptomycin, kanamycin, neomycin, gentamicin, tobramycin, amikacin, etc.): bind irreversibly to 30S ribosomal proteins and inhibit peptide formation; bactericidal.

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.

Resistance to aminoglycosides:

1. Mutation to ribosomal binding site;

2. Decreased uptake of antibiotic;

3. Enzymatic modification of the antibiotic.

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.

Inhibition of nucleic acid synthesisquinolones, rifampin, sulfonamides, trimethoprime, pyrimethamine

AntimetabolitesSulfonamides: 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.

Environment

Amount of pathogen

State of bacterial metabolic activity

Distribution of drug

Location of organisms

Interfering substances

Antimicrobial activity in vivo

Concentration of antibiotic

Absorption

Distribution

Variability of concentration

Factors affecting the effectiveness of antibiotics in vivo

Dangers of indiscriminate use of antibiotics

1. Development of drug resistance.

2. “Superinfection" resulting from changes in the normal f

lora of the body.

3. Masking serious infection without eradicating it.

4. Drug toxicity.

5. Widespread sensitization of the population with resulti

ng hypersensitivity, anaphylaxis, rashes, fever, blood d

isorders, cholestatic hepatitis, and perhaps collagen-v

ascular diseases.

Genetic origin of drug resistance

Chromosomal

Extrachromosomal (e.g., R plasmids)

Can be transferred by conjugation, transformation, and transduction.

1. Give an antimicrobial drug only when it is indicated by

rational choices

2. Give a sufficiently large amount of an effective drug as

early as possible

3. Continue treatment long enough to ensure eradication

of infection

General rules in antimicrobial therapy

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 antibioticsDiagnosisAntibiotic susceptibility tests

Antimicrobial drugs used in combinationIndications

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-I1. 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-II3. 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.  

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.

Sites of Action of Antibacterial Chemical Agents