Transcript Foundations in Microbiology

Talaro Chapter 12
Microbial Control
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Choosing a disinfectant or antiseptic….
often called “germicides”
-gaseous, liquid or solid state
-effective concentration (versus toxicity)
-broad spectrum?
-low toxicity
-penetration of surfaces of inanimate objects or
tissues
-resistance to becoming inactivated by organic
matter
-noncorrosive or nonstaining properties
-odor
-affordability
-availability
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Chemical Decontamination Procedures
3 Levels
1) High - kill endospores… are sterilants –
necessary for medical devices etc. e.g., catheters
(some parts are not autoclavable)
2) Intermediate – Kills fungal spores but not
endospores and generally kills most pathogens –
used to disinfect items that touch mucus
membranes but are not invasive
3) Low – Kills only vegetative cells of bacteria and
fungi… probably kills most pathogens – for
cleaning furniture, straps, electrodes… things
that touch the skin surface
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Chemical Groups of Germicides
1) Halogens (a group of non-metallic
elements– fluorine, bromine, chlorine, and
iodine
Antimicrobial in the non-ionic state…
Fluorine and bromine are dangerous so only
Cl and I are used routinely
Bacteriocidal, bacteriostatic, and even
sporicidal with long contact time
Cl and I are the active ingredients in over 1/3
of all antimicrobials marketed
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Chemical Groups of Germicides (cont.)
Cl: used for almost 200 years
Cl2 gas, OCl- (hypochlorite) and NH2Cl (chloramine)
– all react with water to form hypochlorous acid
(HOCl) – reacts with certain amino acids and
denatures proteins
Disadvantages- relatively unstable… light, alkaline
pH, and organic matter affect stability
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Chemical Groups of Germicides (cont.)
I: Penetrates cells of microorganisms well
2-3% in water or 70% alcohol used a topical
antiseptic for surgery
5-10% for
Iodophores- I complexed to a polymer (e.g.,
polyvinylalcohol)… solves several problems…
-allows for slow release of I
-increases penetration
-less staining and irritating
e.g., Betadine
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Chemical Groups of Germicides (cont.)
2) Phenol derivatives (carbolic acid) – broad
spectrum
-derivatives referred to as phenolics
- toxic to host cells – Lister (antiseptic surgery) –
so not used much as antiseptics – disrupts cell
walls, membranes and denatures enzymes
- all antimicrobials compared to phenol still (the
phenol coefficient)
-phenol used rarely but low percentages in soap
solutions are commonly used (e.g., 1-3%, Lysol)
- hexachlorophene
- triclosan (Safeguard soap)
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Chemical Groups of Germicides (cont.)
3) Chlorhexidine – organic molecule with two
phenol rings and Cl
- targets cell membranes and dentures enzymes
- bacteriocidal for Gram- and Gram+ organisms
but is not sporicidal
- low toxicity – used as obstetric antiseptic
neonatal wash, wound antispetic, mucus
membrane irrigant, preservative in eye solutions,
and handscrubbing for doctors, preparing skin for
surgical procedures
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Chemical Groups of Germicides (cont.)
4) Alcohols - -OH functional groups
- ethyl and isopropyl are the only suitable
candidates
- Concentrations of >50% dissolve lipids
- Dentures proteins as well in cytoplasm
- Optimal concentration is 70% (water is required
for proteins to coagulate)
- Removes oils on skin in which bacteria may be
embedded
- Disadvantages – evaporates quickly, some
organisms can use it, some toxicity with isopropyl
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Chemical Groups of Germicides (cont.)
5) H2O2 and related compounds
- bacteriocidal (broad spectrum) and even
sporicidal at high concentrations
- action is through the hydroxyl free radical (OH-)
- 3% H2O2 (aqueous) is routinely used for skin and
wound cleansing and mouthwashing
- can be a sterilant at 35%... Can get into parts of
medical devices without corrosion
- can be vaporized
- Ozone (O3) can also be used as a disinfectant and
works similarly to preoxide
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Chemical Groups of Germicides (cont.)
6) Detergents and soaps
Detergents – have surface action (called surfactants) – are
polar, charged molecules
- most soaps fit here
- cationic compounds work better than anionic ones… all
have a long uncharged hydrocarbon chain (allows
detergent to disrupt the cell membrane)
- best example – quaternary ammonium compounds
(quats)
- benzalkonium chloride, cetylpyridinium chloride are
commonly used
- mixed with cleaning agents, used to disinfect floors,
furniture, equipment surfaces… not good enough for
medical devices (level of disinfection is low)
- high concentrations – effective against some Gram+,
viruses, fungi, and algae
Low concentrations,
mainly bacteriostatic
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Chemical Groups of Germicides (cont.)
Soaps: made up of fatty acids of oils with sodium or
potassium (are salts of FA)
- Only weakly microbicidal and in fact, many
bacteria including Pseudomonas aeruginosa can
live and grow in soap dishes
- Removes oil on skin however and can be very
effective at removing bacteria on skin if scrubbing
accompanies the washing
- Thus many soaps add some other compound like I
or chlorhexidine
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History of Chemotherapy
• Folk Medicine (pre 1890’s)
• Protonsil Red (1935)
– Plant products
– Streptococci
• Opium/morphine/heroin
– Gerhard Domagk
• Quinine Caffeine Cocaine
– Sulphanomide
• Salicin Digitoxin
• Penicillin (1941)
• Diptheria antitoxin (1891)
– Florey & Chain
• Salvarsan for syphillus (1911)
• Streptomycin (1944)
– Arsenic compound
– Effective against
tuberculosis
– Paul Ehrlich
– Selman Waksman
– “magic bullets”
– Streptococcus griseus
• Penicillium inhibited streptocci on
petri plate (1928)
– Alexander Flemming
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Chemical Groups of Germicides (cont.)
7) Heavy Metals – Hg, Ag, Au, Cu, Zn, As have all been used
at one time or another for microbial control
- Most too toxic to host!!! And can be absorbed through
skin so even tough to use as disinfectants…may cause
allergic reactions
- broad spectrum as they bind and inactivate proteins
- Mercurochrome
- AgNO3 Solutions and newborns – are still some Agcontaining ointments particularly for burns
Some other disadvantages – Microbes can develop resistance
to metals (genes for this often times on the same plasmids
as those that have R factors!)
- Large amounts of organic material (host wastes etc.)
neutarlize action
- Age generally weak as to their antiseptic qualities
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Chemical Groups of Germicides (cont.)
8) The aldehydes – formaldehyde, and
glutaraldehyde (have –CHO functional group)
- Intermediate to high level disinfectants
- “fixes” proteins- preserves tissues for many
years…
- Very toxic
- Glut can be even used as a sterilant… killing
spores in about 3 hours
- “chemiclave” - vaporized glut
- Formaldehyde gas – formalin (37% aqueous
solution) – acts more slowly than glut
- Formalin is a carcinogen
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Chemical Groups of Germicides (cont.)
9) Gaseous sterilants and disinfectants
a) Ethylene oxideb) Propylene oxide –
c) Chlorine dioxide -
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Chemical Groups of Germicides (cont.)
10) Dyes – even bacterial stains
- Crystal violet
-Malachite Green
11) Acids and Bases –
- Acetic acid –
12) Salt (NaCl) -
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Remember:
Antiseptic and Disinfecting
Activity is:
1) Concentration (of compound)
2) Time
3) Organism
Dependent!!!
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Microorganisms & Antimicrobial Drugs
• Antibiotics are common metabolic products of
aerobic bacteria & fungi
– Streptomyces (an Actinomycete) & Bacillus
– Penicillium & Cephalosporium (fungi)
• Inhibit other microbes in the same habitat,
antibiotic producers have less competition for
nutrients & space
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Mechanism of Antimicrobial Action
• Antimicrobial drugs may simply inhibit bacterial growth
BACTERIOSTATIC
• Drugs may also actively kill bacteria - BACTERIOCIDAL
• All exert action by inhibiting particular aspect of cellular
physiology
• Five primary aspects of cellular physiology are targets
• Cell Wall Synthesis
• Protein Synthesis
• DNA/RNA Synthesis
• Maintenance of Plasma Membrane
• Synthesis of Essential Metabolites
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Selective Toxicity
Essential Metabolite
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Inhibition of Cell Wall Synthesis
Peptidoglycan
b-lactam
Weak points in
peptidoglycan
Bulging surface of cocci
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Page 352
b-lactams
BSCI 424 PATHOGENIC MICROBIOLOGY U of Maryland
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N-acetylmuramic acid
(NAM)
Peptidoglycan
• Macromolecule composed of a
repeating framework of long chains
cross-linked by short peptide fragments
– Unique to Bacteria
– Composed of 2 sugars: NAG &
NAM
– Sugars alternate in the backbone
– Rows linked by polypeptides
N-acetylglucosamine
(NAG)
• Provides strong, flexible support to keep
bacteria from bursting or collapsing
because of changes in osmotic pressure
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Intact peptidoglycan
NAM & NAG glycans cross
linked by peptide bridges
Relatively safe since
mammals do not have
peptidoglycan
b-lactam antibiotics block
peptidases that link the cross
bridges between NAMs
Penicillins
Cephalosporins
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Broad spectrum penicillins
and cephalosporins can
cross the cell walls of Gram
negative bacteria.
Carbenicillin & ceftriaxone
Some penicillins are less effective against Gram
negative bacteria.
Some cannot penetrate the outer membrane well.
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Penicillins
• All consist of 3 parts
– Thiazolidine ring
– b-lactam ring
– Variable side chain dictates
microbial activity
•R
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Neisseria gonorrhoeae
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Cephalosporins
• Account for majority of all
antibiotics administered
• Cephalosporium acremonium
• b-lactam
• Relatively broad-spectrum
• Resistant to most penicillinases
& cause fewer allergic reactions
• Some are given orally, many
must be administered
parenterally
– Other than via the digestive tract
• Intravenous or intramuscular
injection
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Vancomycin
• Streptomyces orientalis
• Disrupt alanine-alanine bridges that link
NAM in most Gram + bacteria
– Bacteria lacking alanine-alanine bridges are
resistant
publications.nigms.nih.gov/.../chapter1.html
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Bacitracin
• Peptide antibiotic
– In the product Neosporin
• Bacillus subtilis
• Blocks movement of NAG and NAM from the
cytoplasm
• Effective against Gram +
• Topical antibiotic preparations
– Bacitracin has a high toxicity which precludes its
systemic use
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• b-lactams, vancomycin and bacitracin inhibit
bacteria from building peptidoglycan
• Have no effect on already existing peptidoglycan
• Thus really most effective against actively
growing cells
• Dormant cells or endospores are not susceptible
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Inhibitors of Specialized Cell Wall Synthesis
• Unusual cell walls - Mycobacterium tuberculosis
• Isoniazid inhibits synthesis of mycolic acids… acid-fast stain…
• Integral part of waxy cell wall
• Essential for cell wall assembly
• Ethambutol inhibits incorporation of mycolic acid into waxy layer
• Both extremely effective against Mycobacterium
• Mycobacteria have generation times of 12 – 24 hours (slow growers)
• Drugs administered over many months
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Outer lipids
Lipoarabinomannan
Mycolic acid
Cell wall
skeleton
Arabinogalactan
Peptidoglycan
Plasma membrane
Mycolic acids make up much of the cell wall of Mycobacterium
es.wikipedia.org/wiki/Imagen:Mycobacterial_cell_wall_diagram.png
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Drugs that Block Protein Synthesis
• Ribosomes of eukaryotes differ in size and
structure from prokaryotes
- 80s versus 70s – don’t forget subunit
differences…
– Antimicrobials selectively target bacterial
translation
– Some of these drugs damage ribosomes in the
eukaryotic mitochondria or chloroplast
• Contain a prokaryotic type of ribosome
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Most of These Effect Some Part of Translation
• Aminoglycosides
– Insert on sites on the 30S subunit and interfere with the reading of the
codons on the mRNA
• Tetracyclines
– Block attachment of tRNA on the A acceptor site and stop further synthesis
• Chloramphenicol
– Attach to the 50S subunit
– Prevents formation of peptide bonds
• Macrolides & Lincosamides
– Attach to the 50S subunit
– Inhibit translocation
• Movement of ribosome from one codon to another
• Oxazolidinones
– Inhibits initiation complex formation
– Binds to the 50S subunit and prevents the 30S complex from forming the
70S complex
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Antibiotics That Interfere With Protein
Synthesis
Aminoglycosides – Gram- aerobic bacteria
-Pseudomonas, Enterobacter, even
Mycobacterium…
- not well absorbed through gut so usually
administered intravenously and intramuscularly
Examples: Streptomycin, Neomycin, Gentamycin,
Kanamycin
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Antibiotics That Interfere With Protein
Synthesis
Tetracyclines: - Broad spectrum
- First one was chlortetracycline (aureomycin) by
Streptomyces aureofaciens… then Oxytetracycline
by S. nimosus
- Naturally-occurring versus synthetic
- Generally low toxicity… but Ca2+ problem…
absorbed well though gut
- In 2005 tigecycline was developed… a new class
of antibiotics called the glycylcyclines… first new
tetracycline antibiotic in over 20 years… new ones
are in clinical trials now
- Examples: Synthetic- Doxycycline, Minocycline
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Antibiotics That Interfere With Protein
Synthesis
Macrolides - broad spectrum
- Used for upper resp. and soft tissue
infections
- Used often as substitute for penicillin
- Toxicity low- absorbed well through gut
- Examples- erythromycin, azithromycin
(Zithromax or Z-Pak)
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Antibiotics That Interfere With Protein
Synthesis
Lincosamides – action like macrolides
- Lincomycin from S. lincolnensis
- Used to treat Staph and Strept infections and also
good against some anaerobes, and protozoans such
as Plasmodium (malaria)
- Other examples- Clindamycin (basically replaced
Lincomycin)
- Clindamycin usually give intravenously
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Antibiotics That Interfere With Protein
Synthesis
Oxazolidinones – Good against Gram+’s
- These were developed in the 90’s and used
against MRSA when Vancomycin failed…
- First one developed was Linezolid (Zyvox)…
- Excellent oral bioavailability… and can be
injected as well
- Other examples: AZD2563 (AstraZeneca)…shows
great promise….
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Drugs that Disrupt Cell Membrane Function
• A cell with a damaged membrane dies from disruption in
metabolism or lysis
• These drugs have specificity for a particular microbial
group
– Based on differences in types of lipids in their cell membranes.
• Polymyxins
–
–
–
–
Interact with phospholipids and cause leakage
Originally the only class of antibiotic for Gram - bacteria
Not effective against Gram + bacteria
Topical use
• Toxic to kidney cells
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• Pyrazinamide
– May inhibit fatty acid synthesis
– Transport across the cytoplasmic membrane is
disrupted
– Uniquely effective against Mycobacterium
• Mycobacterium accumulates this drug
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Inhibition of an Essential Metabolite
• Sulfonamides and trimethoprim interfere with folate
metabolism
– Block enzymes required for tetrahydrofolate synthesis needed for DNA
& RNA synthesis
• Competitive inhibition by sulfonamides
– Structural analog
– Drug competes with normal substrate for enzyme’s active site
• para-aminobenzoic acid (PABA)
• Trimethoprim inhibits dihydrofolate reductase
– Next step in the tetrahydrofolate acid biosynthetic pathway
• Synergistic effect
– Both drugs administered together
– An additive effect, achieved by multiple drugs working together,
requiring a lower dose of each
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Sulphonamides inhibit dihydropteroate
synthetase since it is a structural analogue
of the normal substrate, PABA.
Trimethoprim inhibits dihydrofolate
reductase, the next step in the folic acid
biosynthetic pathway.
Humans convert dietary folic acid to THFA
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Quinolones Inhibit DNA Unwinding Enzymes
• Inhibit Gyrase
– Introduces supercoils
• Page 258
– Essential for DNA replication
– Necessary for packaging chromosome such that it
fits inside the cell
• Broad spectrum against Gram + & Gram – Nalidixic acid
– Norfloxacin
– Ciprofloxacin
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Rifampin Inhibits RNA Polymerase
• Rifamycin inhibits prokaryotic RNA polymerase
•
•
•
•
Extremely soluble
Can penetrate many tissues other drugs cannot
Tuberculosis
Meningitis
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Antiviral Agents
• Inhibition of Viral Entry
• Inhibition of Nucleic Acid Synthesis
• Inhibition of Viral Assembly / Release
Prevents binding
Blocks entry
Interferes with neuraminidase
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Inactivates DNA polymerase
Interferes with reverse transcriptase
Interferes with reverse transcriptase
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Interferes with viral protease
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Interferons
• Signaling molecules made by virally infected cells
• Human proteins
• Secreted from infected cell, signals other cells to make anti-viral
proteins (AVP)
• AVP produced in susceptible cells prevent viral replication
• Does not help cells that are already infected
• Only assists uninfected cells in resisting infection
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Mechanisms Drug Resistance
• Drug inactivation
– Penicillinases
– Genes on R plasmids
– 200 different lactamases described
b-lactamase
cephalsporinase
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• Decreased permeability to drug or increased
elimination of drug from cell
– Alteration of shape or charge of porins
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Multidrug resistant pumps
• Self defense ejection system
• Lack selectivity & can pump out
antimicrobials, detergents and toxins
publications.nigms.nih.gov/.../pump_it_up/
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• Change in drug receptors
– Drug cannot bind to target
Vancomycin resistance develops
when one D-alanine is converted
to D-lactate
Instead of 3 H bonds
now only two form
between vancomycin
and D-lactate
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• Change in drug receptor
– Streptomycin resistance
– A missense mutation changes the binding site
on the 30S subunit of bacterial ribosome
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• Change in metabolic patterns
• Produce slightly different enzyme
• Abandon metabolic pathway
• Create a new metabolic pathway
Sulfonamide & trimethoprim resistance
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Preventing Drug Resistance
• Complete the full antimicrobial prescription
– High concentrations of the drug must be maintained for a sufficient time
to eliminate all sensitive cells
• Combinations
– Synergistic
– Some combinations are antagonistic
• Limit use
– Estimated that 50% of the prescriptions for antibacterial drugs to treat
sore throats and 30% of prescriptions for ear infections are unnecessary
• Viral etiology
• Drug Development
– Novel side chains to existing drugs
– New drugs
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• Minimum Inhibitory Concentration (MIC)
– Smallest concentration of drug that visibly inhibits growth
• Therapeutic Index
– The ratio of the dose of the drug that is toxic to humans as
compared to its minimum effective dose
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Disk Diffusion Method
• Spread dilute culture of bacteria over plate
• Place disk containing known amount antibiotic on plate
• Antibiotic diffuses out of disk, creates concentration gradient
• If bacteria is susceptible, a zone of inhibition is produced
• The larger the zone, the more sensitive the organism
• A qualitative test
• Sensitive
• Intermediate
• Resistant
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Larger the zone, the
greater the sensitivity
to the antibiotic
Qualitative test
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Larger the zone, the greater the sensitivity to the antibiotic
(must be comparable so the same antibiotic)
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Quantitative Sensitivity Tests
• E-test is more quantitative
• Spread dilute culture over plate
• Place plastic strip with gradient of
antibiotic
• Zone of inhibition is produced
• Can determine the minimum
inhibitory concentration (MIC) of an
antibiotic required for specific
organism
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Broth Dilution Tests
• More versatile than E-test
• Allows determination of minimal bactericidal concentration (MBC)
as well as MIC
• Make series of dilutions of antibiotic in growth media
• Inoculate each dilution with bacteria
• Determine minimum concentration required to inhibit growth
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But Remember- must reach that concentration in blood
or in specific tissue!!
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