Sterilization, Disinfection and Antibacterial Agents

Sterilization, Disinfection andAntibacterial Agents• Pin Ling ( 凌 斌 ), Ph.D.

Department of Microbiology & Immunology, NCKUext 5632lingpin@mail.ncku.edu.tw

• 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 (bacter

ia, 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)

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 (I)

1. Plasma gas => Hydrogen peroxide Reactive free radicals

=> No Toxic byproducts

=> may replace many applications for ethylene oxide

Cons: NOT good for materials absorbing or reacting with

H2O2

2. Peracetic acid => an oxidizing agent w/ good activity

=> end products nontoxic

3. 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 or

ganisms 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 disinfectantsUsed 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 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

Flaming; incineration

RadiationUV-light: UV-radiation causes damage to DNA.

Ionizing radiation: less applicable.

FiltrationThe 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 agentsFormalin (37% aqueous solution of formaldehyde), glutaraldehydeEthylene 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

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

Outer wall of Gram-positive and Gram-negative species

-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, bacitracin)

PBPs (penicillin-binding proteins): receptors for -lactam drugs. There are 3-6 PBPs, some of which are transpeptidation enzymes.

PenicillinsProduced by Penicillium chrysogenumModifications:

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.

VancomycinA 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

Polymyxins1. 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

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.

Mechanisms

How Bacteria Become Resistant to the -Lactam Antibiotics?1. To prevent the interaction between the antibiotic and the target PB

P e.g. Gram-neg (Pseudomonas) => change porins on the pores => e

xclude 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

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.

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 antibiotics1. 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 therapygive 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 resistance1. 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 combinationIndicationsPrompt 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.

DisadvantagesRelaxation 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)

SUMMARY1. 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 (t

arget) of the drug, or (3) metabolic inactivation of the drug.    4. Combinations of antibiotics may act synergistically-producing an effect stronge

r 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

B. stearothermophilus spores

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Aminoglycosides

Amino sugars

Aminocyclitol

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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.

Sites of antibiotic activity

Disruption of cell wall

Disinfection and SterilizationDisinfection: 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 DNAFormation of pyrimidine dimer by UV irradiationSingle- or double-strand DNA break by ionizing radiation

DNA reactive chemicals, e.g. alkylating agents2. Protein denaturation3. Disruption of cell membrane or wall4. Removal of free sulfhydryl groups

Formation of disulfide bond by oxidizing agentsHeavy 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

: N-acetylglucosamine

: -Ala-IGln-Lys-Ala-

: [Gly]5

Resistance to -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).