Académique Documents
Professionnel Documents
Culture Documents
History of Chemotherapy
Quinine from tree bark was long used to treat malaria. 1910: Paul Ehrlich developed a synthetic arsenic drug, salvarsan, to treat syphilis.
1930s: Sulfonamides synthesized. were
History of Chemotherapy
1928:
Alexander Fleming discovered the first antibiotic. He observed that Penicillium fungus produced an antibiotic, penicillin, that killed S. aureus.
1940s: Penicillin was tested clinically and mass produced.
Antibiotic
Antibiotic
Bacitracin Polymyxin
Cephalothin Penicillin
Mostly targeted against prokaryotic cells Protozoans, fungal and helminthic infections are more difficult to treat
A.
B.
Bactericidal Bacteriostatic
Inhibition of cell wall synthesis Inhibition of protein synthesis Injury to plasma membrane Inhibition of nucleic acid synthesis Antimetabolites
Spectrum of activity
Narrow Broad For gm (+) bacteria For mycolic acid component Broad Broad Broad For gm (-) bacteria For TB infections Broad Broad
Must have a strong activity against the organism Low toxicity to the host Least toxic to normal flora Must have appropriate pharmacological properties Effective blood/tissue levels achieved
Factors to be considered
Host immune status Host organ function Underlying medical disease Age of the patient Site of infection Route of administration Hypersensitivity reaction Crosses placenta?..blood-brain barrier?
Diffusion Methods
Broth Dilution Tests
METHODS
Conventional testing methods:
Broth dilution a. tube (macro-dilution) measures both MIC and MBC b. wells (micro-dilution) measures both MIC and MBC Agar disk diffusion a. Kirby Bauer b. Stokes controlled sensitivity test
Agar cup diffusion employs boring holes on the agar, while streaking of inoculum on agar is done in overlapping manner; antibiotics are in liquid form Agar cylinder makes use of metal or plastic cylinder; antibiotics are poured into hole of cylinder
Alamar system measures MIC; uses a tray that would contain the antibiotics, Mueller Hinton broth, organism plus an Alamar blue indicator. The antibiotics are in decreasing concentration; after incubation there will be a change in color from blue to pink, indicating visible growth
Commercial systems
Reader devices: instrument-assisted reader
TREK diagnostic system- Sensititre Sensitouch SIEMANS- Microscan touchscan
BD Phoenix system: instrument-assisted reader Microscan WalkAway SI TREK Sensititre ARIS 2X VITEK 1, VITEK 2, VITEK 2 Compact
Diffusion Methods
Commonly used in vitro tests in the laboratory
Inexpensive, easy to perform Examples: Kirby Bauer Disk Diffusion Test, Stokes Test
A control organism is inoculated on part of a plate and the test organism is plated on the remainder. Disks are placed at the interface and the zones of inhibition are compared. The use of a sensitive control shows that the antibiotic is active, so that if the test organism grows up to the disk it may safely be assumed that the test organism is resistant to that drug.
*The bacterium in the diagram is susceptible to drug "x" but resistant to drug "y". *The disc containing drug "y" contains active antibiotic as shown by the zone of inhibition it causes in the control bacterium.
Considerations in AST
III. Temperature
IV. Atmospheric condition V. Choice of antibiotic panel VI. Reading and interpretation of results
E TEST
Gradient Strip (AB Biodisk)
E Test
E Test
Dilution Tests
*Tube Dilution
*Agar Dilution MIC and MBC Determinations are done with dilution assays
For microorganisms with unpredictable susceptibilities MIC is the first broth (in tube) in which growth of the organism has been inhibited. The more resistant an organism is, then the higher will be the MIC.
Relatively straightforward
Easy to prepare for and perform Reproducible Can be done on a very small scale (microtiter MIC)
Easy way to test the antimicrobial attributes of a formulation across many different parameters Shorter TAT
Minor variations in MIC test parameters can have major impacts on the apparent MIC Microorganisms were merely prevented from growing-- not necessarily killed: there could still be 500,000 viable cells in that dilution vessel just waiting to grow should the antimicrobial agent become neutralized!
For microorganisms with unpredictable susceptibilities For treatment of serious infections To minimize toxicity effects of drugs
MBC
First dilution at which no growth is observed Cidal drugs have MBC values that are close to the MIC value for particular organisms With static agents, the MIC is much lower than the MBC
The MIC/MBC test of a moderately resistant bacteriostatic drug. Once the bacteria are removed from the drug they can grow on drug free medium at most concentrations.
The MIC/MBC test of a moderately resistant bactericidal drug. Once the bacteria are removed from the drug they cannot grow on drug free medium apart from the tube representing the MIC of the antibiotic.
For mixed infections Prevention of development of bacterial resistance For minimizing toxicity effects When combination therapy would be better option
Terminologies
Autonomous/Indifferent - The result with (2) drugs is equal to the result with the most effective drug by itself Antagonistic - The result with (2) drugs is significantly less than the best individual response
Terminologies
Additive - The result with (2) drugs is equal to the combined action of each of the drug used separately
Synergistic - The result with (2) drugs is significantly better than the additive response
Antimicrobial Resistance
Requires interruption of one or more of the steps essential for effective antimicrobial action Results to partial or complete loss of antibiotic effectiveness
Antimicrobial Resistance
Intrinsic- transmitted vertically to the progeny Acquired- acquisition of mobile genetic elements (transposons, plasmids) capable of disseminating resistant determinants
Antimicrobial Resistance
Intrinsic
Impermeability Biofilms Efflux pumps (transporter CHONs involved in removal of toxic substances from the cell interior to external environment) Enzyme inactivation
Antimicrobial Resistance
Extrinsic
Efflux pump- due to translocation of plasmids Target site modification
Chr mutation Enzymatic target site alteration
Antimicrobial Resistance
Mechanisms:
I.
Destruction or inactivation of the drug Prevention of penetration to target site within the microbe
II.
Antimicrobial Resistance
Rapid efflux, which pumps the drug out of the cell before in can become effective
1. Modification of existing drugs 2. Identifying other targets for antimicrobial activity 3. Development of antimicrobial peptides from other sources
TERM
Minimum Inhibitory Concentration (MIC)
Definition/Comment
Lowest concentration (highest dilution) of antibiotic that inhibits visible growth
Minimum Bactericidal Concentration Lowest concentration (highest dilution) of (MBC) antibiotics that kills 99.9% of the original inoculum
Antimicrobial levels
Serum Bactericidal Level
Synergy test
Good day
ASSIGNMENT
How do you detect antibiotic resistance among bacteria? Gives examples of these (1) detection methods, (2) the microorganism involved/tested, (3) antimicrobial panel used Give examples of methods to detect antimicrobial-inactivating enzymes Discuss the quality control procedures employed when doing antimicrobial susceptibility tests SUBMIT ON FRIDAY Cite your references. Use reliable sources.