Sterilization, Disinfection and

Antibacterial Agents

• Pin Ling ( 凌 斌 ), Ph.D.

Department of Microbiology & Immunology, NCKU
ext 5632
lingpin@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 c
hemical 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 (mi
croplasma)

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 2O2

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 or
ganisms or bacterium spores

2. Categorizing: High-level  sterilization

Intermediate-level Not effective for
all bacteria
Low level or spores

3. Disinfectant: a substance or method used to kill microbe

s 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 spore
s and highly resilient organism, eg. Laryngoscopes, Anesthesia
breathing circuits…etc

Low-level disinfectants
Used to treat noncritical instruments and devices, not penetr
ating 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 7
1.5 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 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 sul
fhydryl group, e.g., peracetic acid, iodine, iodophore, H2
O2 (3-6%), hypochlorite, and chlorine etc.
Plasma gas sterilization: H2O2 vapors treated with micro
wave or radio energy to produce reactive free radicals; n
o toxic byproducts. An efficient sterilization for dry surfac
es.
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.
 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 consist
ing:
a polysaccharide polymer cros
s-linked by Peptide bonds
3. Cross-linking reaction is mediat
ed 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)
-lactam drugs:
Drugs containing a -lactam
ring, e.g. penicillins and cep
halosporins.
Vancomycin: bactericidal for s
ome gram-positive bacteria

PBPs (penicillin-binding proteins): r

eceptors for -lactam drugs. There
are 3-6 PBPs, some of which are tr
anspeptidation enzymes.
 Penicillins
Produced 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 effe
ctiveness.
Modifications of cephalosporins were to expand their
spectra or increase their stability to -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 vanco

mycin

The resistance genes are carried on plasmids

 Polymyxins
1. Cyclic polypeptides (from Bacillus polymyxa)

2. Insert into bacteria outer membrane by interacting w

ith LPS and phospholipids  increase cell perme
ability  bacterial cell death

3. Most Active for gram-negative bacteria, because Gr

am-pos bacteria have no outer member

4. Nephrotoxic

5. External treatment of localized infection and oral ad

ministration 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
-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 penicillins
others have a broad range of activity
 Inhibition of protein synthesis
Aminoglycosides (streptomycin, kanamycin, neomycin, gentamicin, t
obramycin, 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 b
one marrow cells (aplastic anemia); bacteriostatic.
Macrolides (erythromycins, clarithromycin, etc.): bind to 23S rRNA an
d block peptide elongation.
Lincomycins (clindamycin): resembles macrolides in mode of action.
Oxazolidinones (linezolid): blocks formation of imitiation complex. Acti
ve against staphylococci, streptococci and enterococci. No cross-resist
ance with the above antibiotics. Reserved for multidrug-resistant enter
ococci.
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. Red
uction of its nitro group by bacterial nitroreductase produces
cytotoxic compound that disrupts bacterial DNA.

Antimetabolites
Sulfonamides: analogs of p-aminobenzoic acid (PABA) and i
nhibit synthesis of folic acid, which is an important precursor t
o 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 Concentration of
antibiotic
Amount of pathogen
Absorption
State of bacterial
metabolic activity Distribution
Distribution of drug Variability of
concentration
Location of organisms
Interfering substances
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 b
y conjugation, transfo
rmation, and transdu
ction.
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 li
ncomycins)
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 synt
hesis, (4) ribosomal function, and (5) metabolite synthesis.

2. Cell wall synthesis is inhibited by ß-lactams, such as penicillins and cephalosp

orins, which inhibit peptidoglycan polymerization, and by vancomycin, whic
h 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 on
e 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 aci
d 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 polymer
ization, and by vancomycin, which combines with cell wall substrates. Polymyxins disrupt the plasma membrane, causin
g 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, ery
thromycin, and clindamycin all interfere with ribosome function. Sulfonamides and trimethoprim block the synthesis of th
e folate needed for DNA replication       
Bacterial Resistance

Bacteria can evolve resistance to antibiotics. Resistance factors can be encoded on plasmids or on the chromosome. R
esistance may involve decreased entry of the drug, changes in the receptor (target) of the drug, or metabolic inactivatio
n 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 dr
ugs 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 diarrhe
a 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
Back
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 Ger
many, who sought systematically to discover effective agents to treat trypanosomiasis and syphi
lis. He discovered p-rosaniline, which has antitrypanosomal effects, and arsphenamine, which i
s effective against syphilis. Ehrlich postulated that it would be possible to find chemicals that w
ere 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 prot
ective effects of prontosil, the forerunner of sulfonamide. Ironically, penicillin G was discovere
d fortuitously in 1929 by Fleming, who did not initially appreciate the magnitude of his discove
ry. In 1939 Florey and colleagues at Oxford University again isolated penicillin. In 1944 Waks
man isolated streptomycin and subsequently found agents such as chloramphenicol, tetracyclin
es, and erythromycin in soil samples. By the 1960s, improvements in fermentation techniques a
nd advances in medicinal chemistry permitted the synthesis of many new chemotherapeutic age
nts by molecular modification of existing compounds. Progress in the development of novel anti
bacterial agents has been great, but the development of effective, nontoxic antifungal and antivi
ral 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 suc
h 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 me
thods.
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 reactio
n between an enzyme and its substrate.
Peptidoglycan (of Staphylococcus aureus)

: N-acetylmuraminic acid : -Ala-IGln-Lys-Ala-

: N-acetylglucosamine : [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 n
ew PBP
3. Hydrolysis of drug by producing b-lactamase (> 200
different kinds).