sterilization.pdf

STERILIZATION
• All killed
• non-selective
•Sterilized vs not sterilized

Sterilization and disinfection
General bacteriology aconcise course
What is sterilization process?
Sterilization describes a process that destroys or
eliminates all forms of microbial life and is carried out
in health-care facilities by physical or chemical
methods.

In the United States, approximately 46.5 million surgical
procedures and even more invasive medical
procedures—including approximately 5 million
gastrointestinal endoscopies—are performed each
year.

Each procedure involves contact by a medical device or
surgical instrument with a patient’s sterile tissue or
mucous membranes.

A major risk of all such procedures is the introduction
of pathogens that can lead to infection. Failure to
properly disinfect or sterilize equipment carries not
only risk associated with breach of
host barriers
but also risk for person-to-person
transmission
(e.g., hepatitis B virus) and
transmission of environmental
pathogens (e.g., Pseudomonas
aeruginosa).

Sterilization describes a process that destroys or
eliminates all forms of microbial life and is carried out
in health-care facilities by physical or chemical
methods.
Steam under pressure
, dry heat,
EtO gas,
hydrogen peroxide gas
plasma, and liquid chemicals are the principal
sterilizing agents used in health-care facilities

Cleaning is the removal of visible soil (e.g., organic and
inorganic material) from objects and surfaces and
normally is accomplished manually or mechanically
using water with detergents or enzymatic
products.

Thorough cleaning is essential before high-level
disinfection and sterilization because inorganic and
organic materials that remain on the surfaces of
instruments interfere with the effectiveness of these
processes.

Disinfection describes a process that eliminates many
or all pathogenic microorganisms, except bacterial
spores, on inanimate objects

Factors that affect the efficacy of both disinfection and
sterilization include
• prior cleaning of the object
• organic and inorganic load present
• type and level of microbial contamination
concentration of and exposure time to the
germicide physical nature of the object (e.g.,
crevices, hinges, and lumens)
• presence of biofilms
• temperature and pH of the disinfection process;
and in some cases, relative humidity of the
sterilization process (e.g., ethylene oxide).

Main Methods of Sterilization
Physical Methods: ...
•Radiation Method: ...
•Ultrasonic Method: ...
•Chemical Method:

We sterilize
culture media
surgical instruments
critical items
we disinfect
devices
equipenents

A disinfectant is a chemical substance or
compound used to inactivate or
destroy microorganisms on inert surfaces
floor
surfaces of surgical tables

Chlorine
in drinking water some countries
for washing of clothes
in swimming pools
H2o2
contact lenses
OPA
endoscope for 12 minutes

Iodophor
skin disinfectant

An antiseptic is a chemical agent
that slows or stops the growth of
micro-organisms on external
surfaces of the body and helps to
prevent infections

Sterilization
• autoclaving
• 121oC (heat/pressure)
* Heat resistant materials
• ethylene oxide
• Non heat resistant
• usually equipment
• ultra-violet light
• surfaces (e.g operating rooms)
• not totally effective

Membrane filters
pores
bacteria

ANTIBIOTICS
• Selectively toxic for bacteria
– bactericidal (killing)
– bacteriostatic (growth inhibition)
• no harm to patient

Antibiotics work together
with immune system

Minimal inhibitory concentration
• lowest level stopping growth
• e. g. zone of inhibition around a disk impregnated
with antibiotic

• Antibiotics that inhibit cell wall
biosynthesis are bactericidal
• Without cell wall, osmotic pressure
causes bacteria to burst

Peptidoglycan synthesis
Cytoplasm Cell wall
undecaprenol
sugar
amino
acid
Cell Membrane

Cycloserine
1. alanine (ala) analog
2. inhibits conversion of L-ala to D-ala
3. inhibits formation of D-ala-D-ala

Cycloserine
Analog of alanine
X
Cytoplasm
sugar
amino
acid
X
X
X

Bacitracin
• Inhibits dephosphorylation

Antibiotics
• destroy structures
• present in bacteria
• not present in host

Bacitracin
Cell membrane
undecaprenol
P
P

Vancomycin
• binds to D-ala-D-ala
• inhibits cross-linking

Beta lactam antibiotics
• penicillins
• cephalosporins
• monobactams
• inhibit penicillin binding proteins
• stop cross-linking

Beta lactams
Cell wall
Penicillin binding protein

Cross-linking of peptidoglycan

C NH CH CH C
O
O C N CH
CH3
CH3
COOH
S
Site of penicillinase action.
Breakage of the lactam ring.
STRUCTURE OF PENICILLIN

Attached to lactam ring
• penicillins
• 5 membered ring
• cephalosporins
• 6 membered ring
• monobactams
• no second ring

Chemical modifications change biological
activity
• Early lactam antibiotics
• inactive against Gram negative bacteria
• no penetration of outer membrane

Resistance mechansims
• Produce beta lactamase (penicillinase)
• destroys antibiotic
• modified penicillin binding proteins
• don’t bind antibiotic
• modified porins
• no internalization of antibiotic

• beta lactam
• binds strongly beta lactamases
– inhibits activity
Clavulinic acid

Polymyxin B
• binds
– lipid A
– phospholipids
• disrupts outer membrane, Gram negative bacteria
• toxic to human cells

Principles and Definitions
• Selectivity
• Selectivty8 toxicity9
• Therapeutic index
• Toxic dose/ Effective dose
• Categories of antibiotics
• Bactericidal
• Usually antibiotic of choice
• Bacteriostatic
• Duration of treatment sufficient for host defenses

• Selectivity
• Therapeutic index
• Categories of antibiotics
– Use of bacteriostatic vs bactericidal antibiotic
• Therapeutic index better for bacteriostatic antibiotic
• Resistance to bactericidal antibiotic
• Protein toxin mediates disease – use bacteriostatic
protein synthesis inhibitor

• Antibiotic susceptibility testing (in vitro)
• Minimum inhibitory concentration (MIC)
• Lowest concentration that results in inhibition of visible growth
• Minimum bactericidal concentration (MBC)
• Lowest concentration that kills 99.9% of the original inoculum

Antibiotic Susceptibility Testing
8 4 0
2 1
Tetracycline ( g/ml)
MIC = 2 g/ml
Determination of MIC
Chl Amp
Ery
Str
Tet
Disk Diffusion Test

Antimicrobial agent
(amt. per disk)
and organism
Zone diameter (mm) Approx. MIC
(g/ml) for:
R I MS S R S
Ampicillin (10 g)
Enerobacteriacae 11 12-13 14 32 8
Haemophilus spp. 19 20 4 2
Enterococci 16 17 16
Tetracycline (30 g) 14 15-18 19 16 4
Zone Diameter Standards for Disk Diffusion Tests

• Combination therapy
• Prevent emergence of resistant strains
• Temporary treatment until diagnosis is made
• Antibiotic synergism
• Penicillins and aminoglycosides
• CAUTION: Antibiotic antagonism
• Penicillins and bacteriostatic antibiotics
• Antibiotics vs chemotherapeutic agents vs
antimicrobials

Antibiotics that Inhibit Protein Synthesis

Review of Initiation of Protein Synthesis
30S
1 3
2 GTP
1 2 3 GTP
Initiation Factors
mRNA
3
1
2 GTP
30S
Initiation
Complex
f-met-tRNA
Spectinomycin
Aminoglycosides
1
2
GDP + Pi
50S
70S
Initiation
Complex
A
P

Review of Elongation of Protein Synthesis
GTP
A
P
Tu GTP Tu GDP
Ts
Ts
Tu
+
GDP
Ts
Pi
P A
Tetracycline
A
P
Erythromycin
Fusidic Acid
Chloramphenicol
G GTP
G GDP + Pi
G
GDP
A
P
+
GTP

Survey of Antibiotics

Protein Synthesis Inhibitors
• Mostly bacteriostatic
• Selectivity due to differences in prokaryotic and eukaryotic ribosomes
• Some toxicity - eukaryotic 70S ribosomes

Antimicrobials that Bind to the 30S
Ribosomal Subunit

Aminoglycosides (bactericidal)
streptomycin, kanamycin, gentamicin, tobramycin, amikacin,
netilmicin, neomycin (topical)
• Mode of action - The aminoglycosides irreversibly bind to the 16S
ribosomal RNA and freeze the 30S initiation complex (30S-mRNA-tRNA)
so that no further initiation can occur. They also slow down protein
synthesis that has already initiated and induce misreading of the
mRNA. By binding to the 16 S r-RNA the aminoglycosides increase the
affinity of the A site for t-RNA regardless of the anticodon specificity.
May also destabilize bacterial membranes.
• Spectrum of Activity -Many gram-negative and some gram-positive
bacteria; Not useful for anaerobic (oxygen required for uptake of
antibiotic) or intracellular bacteria.
• Resistance - Common
• Synergy - The aminoglycosides synergize with $-lactam antibiotics. The
$-lactams inhibit cell wall synthesis and thereby increase the
permeability of the aminoglycosides.

Tetracyclines (bacteriostatic)
tetracycline, minocycline and doxycycline
• Mode of action - The tetracyclines reversibly bind to the 30S ribosome
and inhibit binding of aminoacyl-t-RNA to the acceptor site on the 70S
ribosome.
• Spectrum of activity - Broad spectrum; Useful against intracellular
bacteria
• Adverse effects - Destruction of normal intestinal flora resulting in
increased secondary infections; staining and impairment of the
structure of bone and teeth.

Spectinomycin (bacteriostatic)
• Mode of action - Spectinomycin reversibly interferes with m-RNA interaction with the 30S
ribosome. It is structurally similar to the aminoglycosides but does not cause misreading of
mRNA.
• Spectrum of activity - Used in the treatment of penicillin-resistant Neisseria gonorrhoeae
• Resistance - Rare in Neisseria gonorrhoeae

Antimicrobials that Bind to the 50S
Ribosomal Subunit

Chloramphenicol, Lincomycin, Clindamycin
(bacteriostatic)
• Mode of action - These antimicrobials bind to the 50S ribosome and inhibit peptidyl transferase
activity.
• Spectrum of activity - Chloramphenicol - Broad range;
Lincomycin and clindamycin - Restricted range
• Adverse effects - Chloramphenicol is toxic (bone marrow suppression) but is used in the
treatment of bacterial meningitis.

Macrolides (bacteriostatic)
erythromycin, clarithromycin, azithromycin, spiramycin
• Mode of action - The macrolides inhibit translocation.
• Spectrum of activity - Gram-positive bacteria, Mycoplasma, Legionella

Antimicrobials that Interfere with
Elongation Factors
Selectivity due to differences in prokaryotic and eukaryotic
elongation factors

Fusidic acid (bacteriostatic)
• Mode of action - Fusidic acid binds to elongation factor G (EF-G) and inhibits release of EF-G from
the EF-G/GDP complex.
• Spectrum of activity - Gram-positive cocci

Inhibitors of Nucleic Acid Synthesis

Inhibitors of RNA Synthesis
Selectivity due to differences between prokaryotic and eukaryotic
RNA polymerase

Rifampin, Rifamycin, Rifampicin, Rifabutin (bactericidal)
• Mode of action - These antimicrobials bind to DNA-dependent RNA polymerase and inhibit
initiation of mRNA synthesis.
• Spectrum of activity - Broad spectrum but is used most commonly in the treatment of
tuberculosis
• Combination therapy - Since resistance is common, rifampin is usually used in combination
therapy.

Inhibitors of DNA Synthesis
Selectivity due to differences between prokaryotic and eukaryotic enzymes

Quinolones (bactericidal)
nalidixic acid, ciprofloxacin, ofloxacin, norfloxacin, levofloxacin, lomefloxacin, sparfloxacin
• Mode of action - These antimicrobials bind to the A subunit of DNA
gyrase (topoisomerase) and prevent supercoiling of DNA, thereby
inhibiting DNA synthesis.
• Spectrum of activity - Gram-positive cocci and urinary tract infections
• Resistance - Common for nalidixic acid; developing for ciprofloxacin

Antimetabolite Antimicrobials

Inhibitors of Folic Acid Synthesis
• Basis of Selectivity
• Review of Folic
Acid Metabolism
p-aminobenzoic acid + Pteridine
Dihydropteroic acid
Dihydrofolic acid
Tetrahydrofolic acid
Pteridine
synthetase
Dihydrofolate
synthetase
Dihydrofolate
reductase
Thymidine
Purines
Methionine
Trimethoprim
Sulfonamides

Sulfonamides, Sulfones (bacteriostatic)
• Mode of action - These antimicrobials are analogues of para-aminobenzoic acid and
competitively inhibit formation of dihydropteroic acid.
• Spectrum of activity - Broad range activity against gram-positive and gram-negative bacteria;
used primarily in urinary tract and Nocardia infections.
• Combination therapy - The sulfonamides are used in combination with trimethoprim; this
combination blocks two distinct steps in folic acid metabolism and prevents the emergence of
resistant strains.

Trimethoprim, Methotrexate, Pyrimethamine (bacteriostatic)
• Mode of action - These antimicrobials binds to dihydrofolate reductase and inhibit formation of
tetrahydrofolic acid.
• Spectrum of activity - Broad range activity against gram-positive and gram-negative bacteria;
used primarily in urinary tract and Nocardia infections.
• Combination therapy - These antimicrobials are used in combination with the sulfonamides; this
combination blocks two distinct steps in folic acid metabolism and prevents the emergence of
resistant strains.

Anti-Mycobacterial Antibiotics

Para-aminosalicylic acid (PSA) (bacteriostatic)
• Mode of action - Similar to sulfonamides
• Spectrum of activity - Specific for Mycobacterium tuberculosis

Dapsone (bacteriostatic)
• Mode of action - Similar to sulfonamides
• Spectrum of activity - Used in treatment of leprosy (Mycobacterium leprae)

Isoniazid (INH) (bacteriostatic )
• Mode of action - Isoniazid inhibits synthesis of mycolic acids.
• Spectrum of activity - Used in treatment of tuberculosis
• Resistance - Has developed

Antimicrobial Drug Resistance
• Clinical resistance
• Resistance can arise by mutation or by gene transfer (e.g. acquisition
of a plasmid)
• Resistance provides a selective advantage
• Resistance can result from single or multiple steps
• Cross resistance vs multiple resistance
• Cross resistance -- Single mechanism-- closely related antibiotics
• Multiple resistance -- Multiple mechanisms -- unrelated antibiotics

Mechanisms
• Altered permeability
• Altered influx
• Gram negative bacteria
• Altered efflux
• tetracycline
• Inactivation
• -lactamse
• Chloramphenicol acetyl transferase

Mechanisms
• Altered target site
• Penicillin binding proteins (penicillins)
• RNA polymerase (rifampin)
• 30S ribosome (streptomycin)
• Replacement of a sensitive pathway
• Acquisition of a resistant enzyme (sulfonamides,
trimethoprim)

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