Transcript 投影片 1

Sterilization, disinfection
and antisepsis
Original methods for disinfection
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BC Yang
In 1866 J lister suggested antiseptic
surgery. His rational was (典型的三段式論證) :
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Putrefaction is caused by microbes
Wound sepsis is a form of putrefaction
Wound sepsis is caused by microbes
Carbolic acid, phenol
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 In the previous year Lister had heard that 'carbolic
acid' was being used to treat sewage in Carlise, and
that fields treated with the affluent were freed of a
parasite causing disease in cattle.
 Lister then began to clean wounds and dress them
using a solution of carbolic acid. He was able to
announce at a British Medical Association meeting, in
1867, that his wards at the Glasgow Royal Infirmary
had remained clear of sepsis for nine months.
 Opposition was great In England and the United States
mainly against Lister's germ theory rather than against
his "carbolic treatment."
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Phenols and phenolics
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Carbolic acid
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Low level disinfetanct
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O-phenylphenol (Lysol)
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Hexachlorophene (Phisohex)
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High level for Staphylococcus sp. and
Streptococcus sp.
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Surgical scrubs
Damage membrane, inactive enzymes, denature
proteins
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Mechanism of antimicrobial action
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Damage to DNA: radiation, UV, DNAreactive agents
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Protein denaturation: Heat, pH, metals
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Disruption of cell membrane or cell wall:
detergents, enzymes
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Chemical antagonism: analogs
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Removal of free sulfhydryl groups:
oxidizing agents
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To die or not to die, that is a
question
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Sepsis: microbes contamination
Asepsis: absence of microbes
contamination
Sterilization: 100% killing
Disinfection: loss the ability to cause
disease
-cide or cidal: killing of microorganism
-static or stasis: inhibit growth or
multiplication of microbes
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Killing is a probability
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Die at a constant rate (logarithmically)
Depend on exposure time and microbial load
 Rare will reach 100% (on purpose and money consuming)
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Parameters of sterilization/disinfection
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Action process
 The rate/kinetics: the death rate of a microbial
population follows a logarithmic path.
 Resistances: pure empirical, varied from
culture condition, nature of the environment
 Control/indicators:
 Living:
 heat : spores, themophilic bacteria
 ethylene oxide and radiation: bacillus subtilits var.
nigar.
 Chemical:succinic anhydride (120oC), sulphur
(115oC); color change; etc..
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Action index
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A spoonful work already well?
(一匙靈)
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Why not LD50?
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Phenol coefficient (PC)
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American official analytical
chemist’s use-dilution method
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Staphylococcus aureus,
pseudomonas aeruginosa,
salmonella choleraesuis
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Filter paper method
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BC Yang
As a matter of temperature
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Heat
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Boiling water
 Moist heat (steam under pressure, autoclave)
 Hot-air sterilization (Oven)
 Pasteurization
 Tyndallization
 Incineration
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Low temperature
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Refrigerator temp (4-7oC)
Deep-freeze (-20oC to -40oC); liquid nitrogen (-196oC)
Lyophilization (desiccation in low temperature)
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Pasteurization
別呆了, 不要喝尚青尚新鮮的牛奶!
• 1909 John Ross
Robertson finances
the installation of
Toronto's first milk
pasteurization plant
in the College Street
hospital, 30 years
before it becomes
mandatory.
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Tyndallization
(Fractional sterilization)
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John Tyndall was a man of
science—draftsman, surveyor,
physics professor, mathematician,
geologist, atmospheric scientist,
public lecturer, and mountaineer.
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For killing both vegetative and
spore forming bacteria at
temperature of 100oC or below for
30 min on the consecutive days
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BC Yang
Filtration
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BC Yang
Filtration
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Liquid
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When subjects are heat-labile
0.22/0.45 mm membrane filter
Air
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Large space
Alternative: electrostatic precipitation
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Particles passed through a high voltage electric field
become charged, and attracted to and held by the
plate of opposite potential (remove ~97% of bacteria)
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Wash away:
Surfactants: breaks oily film on skin
do you really
clean your
hands?
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BC Yang
UV radiation
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Ultraviolet is light
with very high energy
levels and a
wavelength of 200-400
nm.
One of the most
effective wavelengths
for disinfection is that
of 254 nm.
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Alcohols
 High
level disinfectant
 Disrupt
protein, disrupt membranes,
dissolve lipids
 70%
ethanol
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BC Yang
碘酒 Halogens (Iodine)
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Iodine combines with tyrosine & oxidized
SH groups on other amino acids
 Tincture: 1-2% I2 + 2% Na2I in 70%
ethanol (for surgical site disinfection).
 Povidone-iodines: Betadine
 Skin disinfection
 Surgical scrub
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BC Yang
The choices?
It always depends!
 The
materials
 The purpose
 The nature of microbes
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BC Yang
Antibiotics
Antibiotics and vaccines are among the biggest
medical advances since 1000. (Culver Pictures)
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BC Yang
Diagrammatic representation of the results of
treatment related to specific chemotherapy
Patients with normal immunity
and uncomplicated mild to
moderate infections
Patients with serious lifethreatening infections
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A brief history of antibiotics
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1495, mercury to treat syphilis.
1630, quinine (chinchona tree) for malarial fever by South
American Indians.
1889, Buillemin defined antibiosis.
1910, Paul Ehrlich developed arsenical compound (Salvarsan) for
syphilis, term: the chemical knife.
1929, Alexander Fleming found penicillin.
1935, Gerhard Domagk showed the value of sulfonamides.
1940, Ernst Chain and Howard Flory demonstrated the effect of
penicillin.
1940-1970, then searching for new antibiotics (based on killing)
~ recent year: modifying old drugs, finding new discipline in
antibacterial combats
Early time in war: thanks penicillin, we can go home now….
Now a day? Antibiotics resistance
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Thanks to work by Alexander Fleming (18811955), Howard Florey ( 1898-1968) and Ernst
Chain (1906-1979), penicillin was first produced
on a large scale for human use in 1943. At this
time, the development of a pill that could reliably
kill bacteria was a remarkable development and
many lives were saved during World War II
because this medication was available.
A. Fleming
E. Chain
H. Florey
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A tale by A. Fleming
• He took a sample of the
mold from the
contaminated plate. He
found that it was from
the penicillium family,
later specified as
Penicillium notatum.
Fleming presented his
findings in 1929, but they
raised little interest. He
published a report on
penicillin and its
potential uses in the
British Journal of
Experimental Pathology.
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Scenario of penicillin action on E. coli
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3
6
5
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1: ordinary appearance
2-4: globular extrusions emerge
5: rabbit-ear forms
6: Ghost form
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An ideal antibiotics
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Broad-spectrum
Did not induce resistance
Selective toxicity, low side effects
Preserve normal microbial flora
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Susceptibility test
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Tube dilution method
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Minimal inhibitory
concentration (MIC): the
smallest amount of
chemotherapeutic agent
required to inhibit the
growth of organism in
vitro
Disk diffusion method
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Zone of inhibition (ZOI):
the correlation of ZOI
and MIC has been
established by FAD
ETest. This commercially-prepared strip creates
a gradient of antibiotic concentration when
placed on an agar plate
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Guidance of antimicrobial therapy
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Minimum inhibitory concentration: lowest
concentration of antibiotic that inhibits visible
growth
 Minimum bactericidal concentration: lowest
concentration of antibiotic that kills 99.9% of the
inoculum
 Serum bactericidal title: dilution of serum that
kills 99.9% of the inoculum
 Synergy test: synergistic activity of multiple
antibiotics
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BC Yang
In vitro: Factors for optimal antibiotic action
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pH of environment:
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Components of medium:
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Aminoglycosides and chloramphenical are stable for long
period in vivo.
Size of inoculums:
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Anionic detergents inhibit aminoglycosides, serum proteins
bind to penicillin in varying degrees.
Stability of drug:
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Nitrofurantoin is more active in acid pH; sulfonamides and
aminoglycoside are more active in alkaline pH.
The larger the bacterial inoculum, the greater the chance
for resistnat mutant to emerge.
Metablic activity of microorganisms:
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Actively and rapidly growing organisms are more
susceptible to drug action
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BC Yang
Affecting factors in vivo
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Abscess: circulation is blocked off.
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Foreign bodies: obstruction of the
urinary, biliary or respiratory tracts
etc.
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Immunity.
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BC Yang
Sites of action
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BC Yang
Modes of action (1)
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Penicillins, cephalosporin, bacitracin,
carbapenems and vancomycin.
vancomycin
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Tetracyclines
For lecture only
Inhibitors of Cell Membrane.
Polyenes - Amphotericin B, nystatin, and
condicidin.
Imidazole - Miconazole, ketoconazole and
clotrimazole.
Polymixin E and B.
Amphotericin
Aminoglycosides
Inhibitors of cell wall synthesis.
Inhibitors of Protein Synthesis.
Aminoglycosides - Streptomycin, gentamicin,
neomycin and kanamycin.
Tetracyclines - Chlortetracycline,
oxytetracycline, doxycycline and minocycline.
Erythromycin, lincomycin, chloramphenicol
and clindamycin.
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BC Yang
Modes of action (2)
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Inhibitors of metabolites
(Antimetabolites).
Sulfonamides - Sulfanilamide, sulfadiazine silver
and sulfamethoxazole.
Trimethoprim, ethambutol, isoniazid.
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Inhibitors of nucleic acids
(DNA/RNA polymerase).
Quinolones - Nalidixic acid, norfloxacin and
rifamycin
ciprofloxacin.
Rifamycin and flucytosine.
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BC Yang
Penicillin: an extensively
studied example
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BC Yang
Action mechanism of penicillin
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Action target: cell wall
 on penicillin binding proteins (PBPs)
 Transpeptidases (form cross-links in
peptidoglycan)
 Beta-lactam ring attached to 5-membered
thiazolidine ring
Accessibility of PBPs differ in gram+ and grambacteria
Amino acyl side chain groups determine
spectrum, adsorption, susceptibility to
lactamase
Bactericidal inhibitors
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Resistance
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Failure to bind to PBPs
Cannot penetrate porins (gram-)
Production of lactamase (penicillinase)
Lack autolytic enzyme
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B-lactamase
Types:
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Different substrate specificity
Penicillinases
cephalosporinases
Metallo-b-Lactamase
Location:
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Gram+: extracellularly
Gram-: periplasmic space
Serine-b-Lactamase
By Dr. Osnat Herzberg
University of Maryland Biotechnology Institute (UMBI)
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In Rwanda
聯合報 八十五年 四月十三日 頭版新聞 37
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BC Yang
Resistances
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Natural (inherent) resistance
 Structural barrel
 Lack of target
 Transport system
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Acquired resistance
 Mutation
 Gene exchange (conjugation in most)
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Transferable antibiotic
resistance in bacteria
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Reduced uptake into cell (chloramphenicol)
Active efflux from cell (tetracycline)
Modification of antibiotic targets (b-lactam,
erythromycin)
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inactivation of antibiotic by anzymic modification:
hydrolysis (b-lactam, erythromycin); derivatization
(aminoglycosides)
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Sequestration of antibiotic by protein binding (blactam)
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Metabolic bypass (sulfonamides)
Overproduction of antibiotic target (titration:
sulfonamides)
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BC Yang
Some probable overuse/misuse
of antibiotics
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Prophylatic use before surgery
Empiric use (blinded use)
Increased use of broad spectrum agents
Pediatric use for viral infections
Patients who do not complete course
(chronic disease, eg. TB, AIDS)
Antibiotics in animal feeds
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BC Yang
Policy to deal drug resistance (1)
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Ideally, bacteriological management of clinical
infection should involve:
1. Identification of causative organism
2. Sensitivity test
3. Follow-up the drug effect
4. Monitor antibiotic level to avoid toxicity.
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In reality, most patients requiring antimicrobial
therapy are treated empirically. In serious infections
immediate chemotherapy may be life-saving.
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BC Yang
Policy to deal drug resistance (2)
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Periodic changes of antibiotics used might
change selective pressure and thus avoid the
emergence of resistance and retain the
therapeutic value of antibiotics over a longer
period.
The unnecessary prophylactic or animal feeds
use should be discouraged.
Distribution of information on
current/updated infectious microbes (consult
microbiologists): use more targeted
antibiotics
Patient education (不隨便吃藥, 停藥)
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BC Yang