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I. AntimicrobialsII. Antimicrobial Susceptibility TestingHistory of ChemotherapyQuinine from tree bark was long used to treat malaria.

1910: Paul Ehrlich developed a synthetic arsenic drug, salvarsan, to treat syphilis.

1930s: Sulfonamides were synthesized.History of Chemotherapy1928: 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 AntibioticsTreatment with chemicals is chemotherapy.

Chemotherapeutic agents used to treat infectious disease can be synthetic drugs or antibiotics.

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

Examples of Sources of AntibioticsMicroorganismAntibioticGm (+) RodsB. subtilisB. polymyxa

ActinomycetesS. nodosusS. venezuelaeS. griseus

FungiCephalosporium spp.Penicillium notatum

BacitracinPolymyxin

Amphotericin BChloramphenicolStreptomycin

CephalothinPenicillinSpectrum of antimicrobial drugsMostly targeted against prokaryotic cellsProtozoans, fungal and helminthic infections are more difficult to treat

Actions of Antimicrobial DrugsBactericidalBacteriostatic

Actions of Antimicrobial DrugsInhibition of cell wall synthesisInhibition of protein synthesisInjury to plasma membraneInhibition of nucleic acid synthesisAntimetabolites

Commonly Used Antibacterial DrugsMode of actionSpectrum of activity Inhibitors of cell wall synthesisNatural and Semisynthetic PenicillinsCephalosporinsPolypeptidesAnti-TB drugsInhibitors of CHON synthesisChloramphenicolsAminoglycosidesTetracyclinesCause injury to plasma membranePolymyxin BInhibitors of NA synthesisRifamycinQuinolonesAntimetabolitesSulfonamidesNarrowBroadFor gm (+) bacteriaFor mycolic acid component

BroadBroadBroad

For gm (-) bacteria

For TB infectionsBroad

BroadSelection of Antimicrobial AgentsMust have a strong activity against the organismLow toxicity to the hostLeast toxic to normal floraMust have appropriate pharmacological propertiesEffective blood/tissue levels achieved Selection of Antimicrobial agentsFactors to be consideredHost immune statusHost organ functionUnderlying medical diseaseAge of the patientSite of infectionRoute of administrationHypersensitivity reactionCrosses placenta?..blood-brain barrier?

Antimicrobial Susceptibility TestingDiffusion Methods

Broth Dilution Tests

METHODSConventional testing methods:

Broth dilution a. tube (macro-dilution) measures both MIC and MBCb. wells (micro-dilution) measures both MIC and MBCAgar disk diffusion a. Kirby Bauer b. Stokes controlled sensitivity test

Agar cup diffusionemploys boring holes on the agar, while streaking of inoculum on agar is done in overlapping manner; antibiotics are in liquid form

Agar cylindermakes use of metal or plastic cylinder; antibiotics are poured into hole of cylinder

Alamar systemmeasures 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 systemsReader devices: instrument-assisted readerTREK diagnostic system- Sensititre SensitouchSIEMANS- Microscan touchscan BD Phoenix system: instrument-assisted readerMicroscan WalkAway SITREK Sensititre ARIS 2XVITEK 1, VITEK 2, VITEK 2 CompactDiffusion MethodsCommonly used in vitro tests in the laboratory

Inexpensive, easy to perform

Examples: Kirby Bauer Disk Diffusion Test, Stokes TestStokes Controlled Sensitivity TestPrinciple: 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 TestPrinciple:

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 mediuma. pHb. Cation concentrationc. Agar depth

II. Inoculuma. Density *b. Colony appearancec. Growth phase (viability)d. Manner of inoculating onto agar Considerations in ASTIII. 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 TESTGradient Strip(AB Biodisk)E Test

E Test

Dilution Tests*Tube Dilution

*Agar Dilution

MIC and MBC Determinations are done with dilution assaysMinimum 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 DeterminationRelatively straightforward

Easy to prepare for and perform

Reproducible

Can be done on a very small scale (microtiter MIC)

Advantages of MIC DeterminationEasy way to test the antimicrobial attributes of a formulation across many different parameters

Shorter TATDisadvantages of MIC DeterminationMinor 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

MBCFirst 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 MBCStd. 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 combinationsPurpose:

For mixed infections

Prevention of development of bacterial resistance

For minimizing toxicity effects

When combination therapy would be better option

TerminologiesAutonomous/IndifferentThe 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

TerminologiesAdditiveThe 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 responseAntimicrobial ResistanceRequires interruption of one or more of the steps essential for effective antimicrobial action

Results to partial or complete loss of antibiotic effectivenessAntimicrobial ResistanceIntrinsic- transmitted vertically to the progeny

Acquired- acquisition of mobile genetic elements (transposons, plasmids) capable of disseminating resistant determinantsAntimicrobial ResistanceIntrinsicImpermeabilityBiofilmsEfflux pumps (transporter CHONs involved in removal of toxic substances from the cell interior to external environment)Enzyme inactivationAntimicrobial ResistanceExtrinsicEfflux pump- due to translocation of plasmidsTarget site modificationChr mutationEnzymatic target site alterationAcquisition of new targetsEnzymatic inactivation of the drug

Antimicrobial ResistanceMechanisms:

Destruction or inactivation of the drug

Prevention of penetration to target site within the microbeAntimicrobial ResistanceIII. Alteration of drugs target sites

Rapid efflux, which pumps the drug out of the cell before in can become effectiveAddressing Antimicrobial Resistance1. Modification of existing drugs

2. Identifying other targets for antimicrobial activity

3. Development of antimicrobial peptides from other sources

TERMDefinition/CommentMinimum Inhibitory Concentration (MIC)Lowest concentration (highest dilution) of antibiotic that inhibits visible growthMinimum Bactericidal Concentration (MBC)Lowest concentration (highest dilution) of antibiotics that kills 99.9% of the original inoculumAntimicrobial levelsConcentration (ug/ml) of antibiotic in serum (peak and trough)Serum Bactericidal LevelDilution of serum that kills 99.9% of inoculumExample: Schlichter test which requires peak and trough serum samples tested against inoculumSynergy testSynergistic activity of multiple antibiotics; a research procedure for immunosuppressed patientsGood dayASSIGNMENTHow do you detect antibiotic resistance among bacteria?Gives examples of these (1) detection methods, (2) the microorganism involved/tested, (3) antimicrobial panel usedGive examples of methods to detect antimicrobial-inactivating enzymes Discuss the quality control procedures employed when doing antimicrobial susceptibility testsSUBMIT ON FRIDAYCite your references. Use reliable sources.