Sterilization, Disinfection and Antibacterial Agents

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Sterilization, Disinfection andAntibacterial
Agents

• Pin Ling (? ?), Ph.D.
• Department of Microbiology Immunology, NCKU
• ext 5632
• lingpin_at_mail.ncku.edu.tw
• References
• 1. Murray, P. et al., Medical Microbiology, Ch10
  (5th edition)
• 2. Samuel Baron, Medical Microbiology Ch11 (4th
  edition)

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Outline

• Sterilization (Definition Methods)
• Disinfection (Definition Methods)
• Mechanisms of Antimicrobial Action
• Antibacterial Agents

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What is Sterilization?

• Sterilization (in Microbiology)
• To completely remove all kinds of microbes
  (bacteria, mycobacteria, viruses, fungi) by
  physical or chemical methods.
• Effective to kill bacterium spores
• 3. Sterilant material or method used to remove
  or kill all microbes

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Methods of sterilization (I)
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Methods of sterilization (II)
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Pros Cons of Sterilants (I)

• Steam (Moist) Dry Heat gt the most common
  methods for most materials.
• Cons NO good for heat-senstive, toxic or
  volatile chemicals
• Filtration gt 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)
• Ethylene oxide gt the most common gas vapor
  sterilant
• Cons (1) flammable explosive (2) potential
  carcinogenic
• 4. Formaldehyde gas gt carcinogenic

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Pros Cons of Sterilants (II)

• Plasma gas gt Hydrogen peroxide Reactive
  free radicals
• gt No Toxic byproducts
• gt may replace many applications for ethylene
  oxide
• Cons NOT good for materials absorbing or
  reacting with
• H2O2
• Peracetic acid gt an oxidizing agent w/ good
  activity
• gt end products nontoxic
• Glutaraldehyde gt Not safe

Microwave - or radio-freq energy
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What is Disinfection?

• Disinfection (in Microbiology)
• To kill most of microbial forms except some
  resistant organisms or bacterium spores
• 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
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High-level disinfectants Used for items involved
in invasive procedures but NOT withstand
sterilization, e.g. Endoscopes, Surgical
instruments
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Intermediate-level disinfectants Used for
cleaning surface or instruments without bacterial
spores and highly resilient organism, eg.
Laryngoscopes, Anesthesia breathing circuitsetc
Low-level disinfectants Used to treat noncritical
instruments and devices, not penetrating into
mucosa surfaces or sterile tissues
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Considerations of Disinfection

• Effectiveness of disinfectants is influenced by
• Nature of the item to be disinfected
• Number and resilience of the contaminants
• Amount of organic material present
• Type and concentration of disinfectant
• Duration and temperature of exposure

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Antisepsis Antiseptic agents

• Use of chemical agents on skin or living tissues
  to inhibit
• or eliminate microbes
• 2. Antiseptic agents are selected for their
  safety efficacy

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Outline

• Introduction
• Sterilization (Definition Methods)
• Disinfection (Definition Methods)
• Mechanisms of Antimicrobial Action

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Physical methods(moist heat, dry heat,
filtration, radiation)

• Moist heat
• Boiling boiling for 10 min gt Kill most
  vegetative forms of bacteria
• Longer time gt Kill spores
• Addition of 2 Na2CO3 or 0.1 NaOH gt 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 gt
  Kill both the vegetative
• and spore forms of the
  bacteria.
• gt Use Bacillius stearothermophilus spores to
  monitor the effectiveness of Autoclave

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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

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Chemical methods

• 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.

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• Alkylating agents
• 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.
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Outline

• Introduction
• Sterilization (Definition Methods)
• Disinfection (Definition Methods)
• Mechanisms of Antimicrobial Action
• Antibacterial Agents

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The Discovery of Antibacterial Agents

• In 1930s Gerhard Domagk discovered the
  anti-bacterial effect of prontosil (gt
  sulfanilamide) gt 1939 Nobel Laureate
• A. Fleming discovered that the mold Penicillium
  prevented the multiplication of staphyloocci.
• gt The first antibiotic, Penicillin, was
  identified gt 1945 Nobel Laureate
• Streptomycin, tetracyclines others were
  thereafter developed to treat infectious
  diseases.
• Bacteria start developing resistance to these
  agents

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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)
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Sites of Action of Antibacterial Chemical Agents
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• Peptidoglycan
• A major component of cell wall
• Forms a meshlike layer consisting
• a polysaccharide polymer cross-linked by Peptide
  bonds
• Cross-linking reaction is mediated by
• transpeptidases
• DD-carboxypeptidases
• Targets of Penicillin

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Outer wall of Gram-positive and Gram-negative
species
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Inhibition of cell wall synthesis(penicillins,
cephalosporins, vancomycin, cycloserine,
bacitracin)
b-lactam drugs Drugs containing a b-lactam
ring, e.g. penicillins and cephalosporins. Vancomy
cin 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.
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Penicillins Produced by Penicillium
chrysogenum Modifications decrease acid
lability increase absorption 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.
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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
vancomycin The resistance genes are carried on
plasmids
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Polymyxins 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 4.
Nephrotoxic 5. External treatment of localized
infection and oral administration to sterilize
the gut
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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.
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How Bacteria Become Resistant to the b-Lactam
Antibiotics?

• 1. To prevent the interaction between the
  antibiotic and the target PBP
• e.g. Gram-neg (Pseudomonas) gt change porins
  on the pores gt 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 b-lactamases
• - They are in the same family of PBPs
• - Over 200 different b-lactamases
• some are specific for penicillins
• others have a broad range of activity

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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.
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• Resistance to aminoglycosides
• Mutation to ribosomal binding site
• Decreased uptake of antibiotic
• Enzymatic modification of the antibiotic.

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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.
Antimetabolites 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.
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Antimicrobial activity in vivo
Factors affecting the effectiveness of
antibiotics in vivo
Concentration of antibiotic Absorption Distributio
n Variability of concentration
Environment Amount of pathogen State of bacterial
metabolic activity Distribution of drug Location
of organisms Interfering substances
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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.
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Genetic origin of drug resistance
Chromosomal Extrachromosomal (e.g., R
plasmids) Can be transferred by conjugation,
transformation, and transduction.
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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.
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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 Diagnosis Antibiotic
susceptibility tests
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Antimicrobial drugs used in combination Indication
s 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).
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Effects of combined usage of two antibiotics
Indifference (A BA or B) Addition (A
BA B) Synergism (A BA x B)
Antagonism (A B 0 or less)
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SUMMARY
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.  
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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 Combination
s 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.
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Antimicrobial chemoprophylaxis In persons of
normal susceptibility exposed to a specific
pathogen In persons of increased
susceptibility In surgery
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B. stearothermophilus spores
Back
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Back
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Aminoglycosides
Amino sugars
Aminocyclitol
Back
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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.
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Disruption of cell wall
Sites of antibiotic activity
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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.

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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.

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Peptidoglycan (of Staphylococcus aureus)
N-acetylmuraminic acid
-Ala-IGln-Lys-Ala-
Gly5

N-acetylglucosamine
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• Resistance to b-lactam antibiotics
• Prevention of interaction of drug and the target
  PBP
• 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 (gt
  200 different kinds).