Antibiotic Sensitivity Testing PDF

**[Antibiotic Sensitivity Testing]** **Attributes of an Ideal Antimicrobial Agent** - Selective toxicity - What ideal antimicrobial agents exhibit (drug is harmful to a pathogen without being harmful to the host) - Relative rather than absolute (dug in a concentration toler...

**[Antibiotic Sensitivity Testing]** **Attributes of an Ideal Antimicrobial Agent** - Selective toxicity - What ideal antimicrobial agents exhibit (drug is harmful to a pathogen without being harmful to the host) - Relative rather than absolute (dug in a concentration tolerated by the host may damage an infecting microorganism) - Targets: - Receptors not found in host - Biochemical pathways not essential for host - Soluble in bodily fluids - Non-allergenic - Reasonable half-life - Long shelf life - Inexpensive **Side Effects** - Toxicity - Drugs may be toxic to kidneys, liver, or nerves - Considerations needed when prescribing drugs to pregnant women - Disruption of normal microbiota **Range of Effectiveness of Antibiotics** - Broad spectrum - Works on a wide range of bacteria and pathogens - More likely to disrupt microbiota or off target effects - Narrow spectrum - Increased selection, reduce non-selective toxicity - More information needed **Antibiotics Mechanism of Action** +-----------------------------------+-----------------------------------+ | Cell component | Inhibitor of synthesis | +===================================+===================================+ | Cell wall | Beta-lactams | | | | | | - Penicillins | | | | | | - Cephalosporins | | | | | | Other | | | | | | - Vancomycin | +-----------------------------------+-----------------------------------+ | Protein | Anti-50S | | | | | | - Inhibits peptide chain | | | elongation during protein | | | synthesis | | | | | | - Bind 23S rRNA subunit | | | | | | Anti-30S | | | | | | - Interfere directly and | | | causing misreading of mRNA | +-----------------------------------+-----------------------------------+ | Nucleic acid | DNA | | | | | | - Inhibit DNA gyrase and | | | topoisomerase, blocking DNA | | | replication | | | | | | RNA | | | | | | - Interferes with bacterial | | | DNA-dependent RNA polymerase | +-----------------------------------+-----------------------------------+ | Cell membrane | Example: Polymyxin B | | | | | | - Binds to plasma membrane and | | | increases its permeability, | | | while disrupting its | | | structure | +-----------------------------------+-----------------------------------+ | Metabolites | Antimetabolite antibiotics | | | | | | - Inhibitors of folic acid | | | synthesis | | | | | | - Important for many | | | functions including | | | growth and RNA/DNA | | | synthesis | | | | | | - Examples: sulfonamides & | | | trimethoprim | | | | | | - Inhibitors of mycolic acid | | | synthesis | | | | | | - Specific for mycobacteria | | | as it is the major lipid | | | component of their cell | | | walls | | | | | | - Example: isoniazid | +-----------------------------------+-----------------------------------+ **Goals of Antibiotics** - Bactericidal - Kills bacteria - Can be bacteriostatic at low concentrations - Bacteriostatic - Stops bacterial growth - If removed bacteria can continue to grow - Requires the hosts immune system to kill the bacteria **Measurement of Antimicrobial Activity** - Conventional susceptibility testing: +-----------------------------------+-----------------------------------+ | Broth Dilution Testing | - Range of concs for each | | | antibiotic is a series of | | | doubling dilutions | | | | | | - Concentration range of drugs | | | depends on specific criteria | | | | | | - The MICs are then translated | | | into: | | | | | | - Susceptible, | | | intermediate, or | | | resistant (based on | | | correlation between MIC | | | with serum-achievable | | | levels) | | | | | | - Types: macrodilutions (2 mL) | | | and microdilutions (100 uL) | | | | | | Advantages | | | | | | - Quantitative method | | | | | | - Straightforward | | | | | | - Opportunity for automation | | | | | | - Flexible | | | | | | Disadvantages | | | | | | - Time consuming and tedious | | | | | | - Media usage | | | | | | - Possibility of error | +===================================+===================================+ | Agar Dilution Testing | - Serial dilutions of the | | | antibiotic are added to | | | melted solid media, poured | | | and left to cool and solidify | | | | | | - Involves a series of agar | | | plates each containing a | | | different concentration of | | | antibiotic | | | | | | - Then a fixed number of | | | organisms is inoculated on | | | the surface of agar plates, | | | and examined by colony count | | | after incubation | | | | | | Advantages | | | | | | - More gradual changes | | | | | | Disadvantages | | | | | | - Labor intensive | | | | | | - Expensive | | | | | | - Space inefficient | +-----------------------------------+-----------------------------------+ | Disc Diffusion Method | - Bacterial suspension should | | | be standardized | | | | | | - Concentration of antibiotics | | | in the disk should be | | | standardized (provided from | | | the manufacturer) | | | | | | - Incubation time and | | | temperature should be | | | standardized | | | | | | - Standardized agar medium | | | (most used is Mueller-Hinton | | | agar) | | | | | | Protocol | | | | | | 1. Make bacterial lawn | | | | | | 2. Dispense antibiotic disks | | | | | | 3. Press GENTLY with forceps for | | | disks to adhere. | | | Alcohol-flame forceps to | | | sterilize | | | | | | 4. Label plates on the edges | | | | | | 5. Invert plates and incubate | | | | | | Zone of Inhibition | | | | | | - Measure from the center of | | | the disk to a point on the | | | circumference of the zone, | | | multiply by 2 | | | | | | Advantages | | | | | | - Simple, flexible, reduced | | | labour, inexpensive, | | | consistent | | | | | | Disadvantages | | | | | | - Diameter instead of | | | concentration requires | | | interpretation | | | | | | - Qualitative | | | | | | - Limited antibiotics | +-----------------------------------+-----------------------------------+ | Epsilometer Test (E-test) | Advantages | | | | | | - Potentially more consistent | | | and accurate | | | | | | - Relatively easy | | | | | | - Well controlled antibiotic | | | concentrations | | | | | | Disadvantages | | | | | | - Higher costs (\~10x cost of | | | discs) | | | | | | - Limited number of antibiotics | +-----------------------------------+-----------------------------------+ - Automated antimicrobial susceptibility testing - Terms - **Minimum inhibitory concentration** (MIC): the lowest antimicrobial concentration that completely inhibits visible growth of an organism - **Minimum bactericidal concentration** (MBC): the lowest antimicrobial concentration required to kill a particular bacterium **Determining Minimum Bactericidal and Inhibitory Concentrations** A diagram of different types of liquid Description automatically generated **Standardization** - The potential influence of environmental factors on antibiotic activity should be minimized - Conditions for susceptibility testing are standardized in the following reasons: - Optimized growth conditions inhibition of growth is due to antimicrobial agent - Optimized integrity of antimicrobial failure to inhibit growth is the organisms' resistance mechanisms, not environment inactivation - Reproducibility and consistency in the resistance profile **Standardized Components of Antimicrobial Susceptibility Testing** - Time 16 hours - Concentrations of antimicrobial 2-fold dilution series - Total volume 2 mL for microdilution; 100 uL for microdilution - Initial inoculum concentration 0.5 McFarland scale or 0.08-0.12 at 625 nm == 1 x 10^5^ CFU/mL **Limitations of Standardization** - In vivo environment cannot be reproduced, where the actual interaction between the bacteria and antibiotic will take place - Other factors - Antibiotic diffusion into tissues and host cells - Serum protein binding of antimicrobial agents - Status of patient immune system - Virulence of the infecting bacterium **Automated Antimicrobial Susceptibility Testing** - Automatically generates a growth curve based on readings - Algorithm-derived MIC +-----------------------------------+-----------------------------------+ | Test | Description | +===================================+===================================+ | Vivtek System | - Micro plate 'card' with | | | preloaded antibiotics | | | | | | - Photometric measurements | | | | | | - Tests against a standard to | | | reach appropriate turbidity | +-----------------------------------+-----------------------------------+ | MicroScan | - Standard micro-dilution test | | | trays | | | | | | - Fluorometric measurements | +-----------------------------------+-----------------------------------+ | BD Phoenix | - Oxidation-reduction detector | | | and a turbidity measurement | +-----------------------------------+-----------------------------------+ - Advantages - Less tedious - More reproducible - Fast - Automated data output - Disadvantages - Limited types of antimicrobials - Limited ability to detect some forms of antimicrobial resistance - Expensive **How are breakpoints calculated/selected?** - All testing requires breakpoints (AKA interpretive criteria), so that the results of the tests can be interpreted and reported as: +-----------------------------------+-----------------------------------+ | Susceptible | Indicates that the antimicrobial | | | agent may be an appropriate | | | choice (in this | | | | | | situation). Bacterial resistance | | | is absent or clinically | | | insignificant. | +===================================+===================================+ | Intermediate | Indicates a number of | | | possibilities, including: | | | | | | - The potential utility of the | | | agent in the body sites where | | | it may be concentrated | | | | | | - The response rates may be | | | lower than those susceptible | | | isolates | | | | | | - Providing a "buffer" between | | | the resistant and susceptible | | | categories to prevent serious | | | interpretive errors | +-----------------------------------+-----------------------------------+ | Resistant | Indicates that the isolates are | | | not inhibited by the usually | | | achievable concentrations of the | | | agent with normal dosage | | | schedules and/or demonstrates | | | that the MICs/Zone diameters that | | | fall in the range where the | | | specific microbial resistance | | | mechanisms are likely and that | | | clinical efficacy against the | | | isolates has not been shown | | | reliably in treatment studies | +-----------------------------------+-----------------------------------+ - Depending on the method, breakpoints are expressed as either a concentration or a zone diameter **"Scattergram" of MICs vs Zone Diameters to Determine Breakpoint for Zone Diameter** - Horizontal lines are drawn from the MIC resistant breakpoint and the susceptible MIC breakpoint - Where the horizontal lines intersect the regression line, vertical lines are drawn to delineate the corresponding inhibitory zone size breakpoints in mm **Breakpoints Categories** - In the category intermediate, has multiple purposes: - "Strains with MICs in the given range is too small to derive robust conclusions" - Responses can be variable - May be dose dependent - Fills in the grey area between resistant and susceptible **Selection of Antimicrobial Agents for Lab Testing** - Agents chosen for testing are antimicrobial batteries or panels - Content and application of each battery based on: - Organisms' identification or group - Acquired resistance patterns common to local microbial flora - Site of infection **Dangers of Indiscriminate Use** - Indications for administration of antibiotic must be qualified by concerns: - Changes in the normal flora of the body, with disease resulting from "superinfection" due to overgrowth of drug-resistant organisms - Direct drug toxicity - Development of drug resistance in microbial populations, chiefly through the elimination of drug-sensitive microorganisms from antibiotic-saturated environments and their replacement by drug-resistant microorganisms **All antibiotic orders must have:** - When to take them - How much to take How many days you should take them