I. Antimicrobials II.

Antimicrobial Susceptibility Testing

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.

Development and Use of Antibiotics

Treatment with chemicals is chemotherapy. Chemotherapeutic agents used to treat infectious disease can be synthetic drugs or antibiotics.

Antibiotic

A substance produced by microorganisms that, in small amounts, inhibits another microorganism

Examples of Sources of Antibiotics

Microorganism
Gm (+) Rods B. subtilis B. polymyxa Actinomycetes S. nodosus S. venezuelae S. griseus Fungi Cephalosporium spp. Penicillium notatum

Antibiotic
Bacitracin Polymyxin

Amphotericin B Chloramphenicol Streptomycin

Cephalothin Penicillin

Spectrum of antimicrobial drugs

Mostly targeted against prokaryotic cells Protozoans, fungal and helminthic infections are more difficult to treat

Actions of Antimicrobial Drugs

A.
B.

Bactericidal Bacteriostatic

Actions of Antimicrobial Drugs

Inhibition of cell wall synthesis Inhibition of protein synthesis Injury to plasma membrane Inhibition of nucleic acid synthesis Antimetabolites

Commonly Used Antibacterial Drugs

Mode of action
Inhibitors of cell wall synthesis Natural and Semisynthetic Penicillins Cephalosporins Polypeptides Anti-TB drugs Inhibitors of CHON synthesis Chloramphenicols Aminoglycosides Tetracyclines Cause injury to plasma membrane Polymyxin B Inhibitors of NA synthesis Rifamycin Quinolones Antimetabolites Sulfonamides

Spectrum of activity
Narrow Broad For gm (+) bacteria For mycolic acid component Broad Broad Broad For gm (-) bacteria For TB infections Broad Broad

SELECTION OF ANTIMICROBIAL AGENTS

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

SELECTION OF ANTIMICROBIAL AGENTS

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?

Antimicrobial Susceptibility Testing

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

Stokes Controlled Sensitivity Test

Principle:

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.

Stokes Controlled Sensitivity Test

*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.

Kirby Bauer Disk Diffusion Test

Principle: A standardized suspension of organisms is inoculated onto Mueller-Hinton agar. Paper disks impregnated with specific antibiotic concentrations are placed into the agar. After 18-24 hours of incubation, diameters of zones of inhibition are measured. Results are compared with established values to determine the organisms susceptibility or resistance to each antibiotic.

Considerations in Anti-microbial Susceptibility Testing (AST)

I. Growth medium a. pH b. Cation concentration c. Agar depth II. Inoculum a. Density * b. Colony appearance c. Growth phase (viability) d. Manner of inoculating onto agar

Considerations in AST
III. Temperature
IV. Atmospheric condition V. Choice of antibiotic panel VI. Reading and interpretation of results

Inoculating on Mueller Hinton Agar

Disk Placement and Zone Measurement

Sample Results of Disk Diffusion Assay

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

Minimum Inhibitory Concentration (MIC)

Purpose:

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.

Advantages of MIC Determination

Relatively straightforward
Easy to prepare for and perform Reproducible Can be done on a very small scale (microtiter MIC)

Advantages of MIC Determination

Easy way to test the antimicrobial attributes of a formulation across many different parameters Shorter TAT

Disadvantages of MIC Determination

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!

Minimum Bactericidal Concentration (MBC)

Purpose:

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

Std. Concentration Values for MIC Testing

Tube Dilution Assay

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.

Testing of antibiotic combinations

Purpose:

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

Acquisition of new targets Enzymatic inactivation of the drug

Antimicrobial Resistance
Mechanisms:
I.

Destruction or inactivation of the drug Prevention of penetration to target site within the microbe

II.

Antimicrobial Resistance

III. Alteration of drugs target sites

IV.

Rapid efflux, which pumps the drug out of the cell before in can become effective

Addressing Antimicrobial Resistance

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

Concentration (ug/ml) of antibiotic in serum (peak and trough)

Dilution of serum that kills 99.9% of inoculum Example: Schlichter test which requires peak and trough serum samples tested against inoculum Synergistic activity of multiple antibiotics; a research procedure for immunosuppressed patients

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.