Sterilization, Disinfection and Antibacterial Agents

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Sterilization, Disinfection and Antibacterial
Agents

• Pin Lin (? ?), Ph.D.
• Departg ment of Microbiology Immunology, NCKU
• ext 5632
• lingpin_at_mail.ncku.edu.tw
• References
• 1. Chapters 10 20 in Medical Microbiology
  (Murray,
• P. R. et al 5th edition)
• 2. ?????? (??? ???, 4th edition)

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Outline

1. Sterilization (Definition Methods)
2. Disinfection (Definition Methods)
3. Mechanisms of Antimicrobial Action
4. 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)
Sterilize instruments
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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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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)
5. Plasma gas gt Hydrogen peroxide gt 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 6. Peracetic acid gt an
oxidizing agent w/ good activity gt
end products nontoxic 7. 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
non-critical 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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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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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
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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
• gt 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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Outline

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

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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
• 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.

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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
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-I
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.
      
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SUMMARY-II
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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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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Disruption of cell wall
Sites of antibiotic activity
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