1 Sterilization, disinfection and antisepsis BC Yang Original methods for disinfection For lecture only

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Sterilization, disinfection and antisepsis

BC Yang

Original methods for disinfection

For lecture only

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Carbolic acid, phenol

In 1866 J lister suggested antiseptic surgery. His rational was ( 典型的三段式論證 ) :

Putrefaction is caused by microbes Wound sepsis is a form of putrefaction Wound sepsis is caused by microbes

BC YangFor lecture only

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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 Carbolic acid Low level disinfetanct O-phenylphenol (Lysol) Hexachlorophene (Phisohex)

High level for Staphylococcus sp. and

Streptococcus sp. Surgical scrubs

Damage membrane, inactive enzymes, denature

proteins


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Mechanism of antimicrobial action

Damage to DNA: radiation, UV, DNA-reactive agents

Protein denaturation: Heat, pH, metals Disruption of cell membrane or cell wall:

detergents, enzymes Chemical antagonism: analogs Removal of free sulfhydryl groups:

oxidizing agents


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To die or not to die, that is a question

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

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

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

A spoonful work already well?

( 一匙靈 )

Why not LD50?

Phenol coefficient (PC) American official analytical

chemist’s use-dilution method Staphylococcus aureus,

pseudomonas aeruginosa,

salmonella choleraesuis Filter paper method


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As a matter of temperature Heat

Boiling water Moist heat (steam under pressure, autoclave) Hot-air sterilization (Oven) Pasteurization Tyndallization Incineration

Low temperature Refrigerator temp (4-7oC) Deep-freeze (-20oC to -40oC); liquid nitrogen (-196oC) Lyophilization (desiccation in low temperature)

For lecture only BC Yang

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• 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.

Pasteurization別呆了 , 不要喝尚青尚新鮮的牛奶 !


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Tyndallization (Fractional sterilization)

John Tyndall was a man of science

—draftsman, surveyor, physics

professor, mathematician,

geologist, atmospheric scientist,

public lecturer, and mountaineer.

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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Filtration


http://www.minntech.com/images/fiber_bg.jpg

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Filtration Liquid

When subjects are heat-labile 0.22/0.45 mm membrane filter

Air Large space

Alternative: electrostatic precipitation 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: do you really

clean your hands?Surfactants: breaks oily film on skin


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UV radiation 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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碘酒 Halogens (Iodine)

BC Yang

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

For lecture only

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The choices?It always depends!

The materials The purpose The nature of microbes


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Antibiotics

BC Yang

Antibiotics and vaccines are among the biggest medical advances since 1000. (Culver Pictures)

For lecture only

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Diagrammatic representation of the results of treatment related to specific chemotherapy

Patients with normal immunity and uncomplicated mild to moderate infections

Patients with serious life-threatening infections


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A brief history of antibiotics 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 (1881-1955), 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.

E. Chain H. FloreyA. Fleming


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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

1 2 3

456

1: ordinary appearance 2-4: globular extrusions emerge5: rabbit-ear forms6: Ghost form


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An ideal antibiotics

Broad-spectrum Did not induce resistance Selective toxicity, low side effects Preserve normal microbial flora


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Susceptibility test Tube dilution method

Minimal inhibitory concentration (MIC): the smallest amount of chemotherapeutic agent required to inhibit the growth of organism in vitro

Disk diffusion method 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

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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In vitro: Factors for optimal antibiotic action

pH of environment: Nitrofurantoin is more active in acid pH; sulfonamides and

aminoglycoside are more active in alkaline pH. Components of medium:

Anionic detergents inhibit aminoglycosides, serum proteins bind to penicillin in varying degrees.

Stability of drug: Aminoglycosides and chloramphenical are stable for long

period in vivo. Size of inoculums:

The larger the bacterial inoculum, the greater the chance for resistnat mutant to emerge.

Metablic activity of microorganisms: Actively and rapidly growing organisms are more

susceptible to drug action


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Affecting factors in vivo

Abscess: circulation is blocked off.

Foreign bodies: obstruction of the

urinary, biliary or respiratory tracts

etc.

Immunity.


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Sites of action


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Modes of action (1) Inhibitors of cell wall synthesis.

Penicillins, cephalosporin, bacitracin, carbapenems and vancomycin.

Inhibitors of Cell Membrane.Polyenes - Amphotericin B, nystatin, and condicidin.Imidazole - Miconazole, ketoconazole and clotrimazole.Polymixin E and B.

Inhibitors of Protein Synthesis.Aminoglycosides - Streptomycin, gentamicin, neomycin and kanamycin.Tetracyclines - Chlortetracycline, oxytetracycline, doxycycline and minocycline.Erythromycin, lincomycin, chloramphenicol and clindamycin.

Amphotericin

Tetracyclines

Aminoglycosides

vancomycin


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Modes of action (2)

Inhibitors of metabolites

(Antimetabolites).Sulfonamides - Sulfanilamide, sulfadiazine silver

and sulfamethoxazole.

Trimethoprim, ethambutol, isoniazid.

Inhibitors of nucleic acids

(DNA/RNA polymerase).Quinolones - Nalidixic acid, norfloxacin and

ciprofloxacin.

Rifamycin and flucytosine.

rifamycin


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Penicillin: an extensively studied example


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Action mechanism of penicillin

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

gram- bacteria Amino acyl side chain groups determine

spectrum, adsorption, susceptibility to lactamase

Bactericidal inhibitors


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Resistance

B-lactamaseTypes:

Different substrate specificity Penicillinases cephalosporinases

Location: Gram+: extracellularly Gram-: periplasmic space

Serine--Lactamase

Metallo--Lactamase

By Dr. Osnat HerzbergUniversity of Maryland Biotechnology Institute (UMBI)

Failure to bind to PBPs Cannot penetrate porins (gram-) Production of lactamase (penicillinase) Lack autolytic enzyme


http://www.nist.gov/cgi-bin/exit_nist.cgi?url=http://www.umbi.umd.edu/

http://www.cstl.nist.gov/div831/carb/osnat/lactamase.jpg

http://www.cstl.nist.gov/div831/carb/osnat/r3d_5d.jpg

37聯合報八十五年四月十三日頭版新聞

In Rwanda


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Resistances

Natural (inherent) resistanceStructural barrelLack of targetTransport system

Acquired resistanceMutationGene exchange (conjugation in most)


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Transferable antibiotic resistance in bacteria

Reduced uptake into cell (chloramphenicol) Active efflux from cell (tetracycline) Modification of antibiotic targets (-lactam,

erythromycin) inactivation of antibiotic by anzymic

modification: hydrolysis (-lactam, erythromycin); derivatization (aminoglycosides)

Sequestration of antibiotic by protein binding (-lactam)

Metabolic bypass (sulfonamides) Overproduction of antibiotic target (titration:

sulfonamides)


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Some probable overuse/misuse of antibiotics

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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Policy to deal drug resistance (1)

Ideally, bacteriological management of clinical infection should involve:

1. Identification of causative organism2. Sensitivity test3. Follow-up the drug effect4. Monitor antibiotic level to avoid toxicity.

In reality, most patients requiring antimicrobial therapy are treated empirically. In serious infections immediate chemotherapy may be life-saving.


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Policy to deal drug resistance (2) 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 )


Documents

1 Sterilization, disinfection and antisepsis BC Yang Original methods for disinfection For lecture only