AST Exam Flashcards by heather wade

Resistance from genetically encoded traits; can be Intrinsic or Acquired

Microorganism mediated resistance

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Resistance resulting from physical or chemical characteristics of the environment

Environmentally mediated resistance

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Change such that a drug is no longer suitable for clinical use

Clinical resistance

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Change in cell physiology or structure that result in observably reduced susceptibility to an antibiotic (acquired)

Biologic resistance

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Resulting from structural or physiologic state of a microorganism; Present long before antibiotic usage by humans

Intrinsic Resistance

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Concern for infection control; Due to modification of genetic makeup of organism; Organism develops resistance to antibiotic to which it was previously susceptible

Acquired Resistance

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What may cause acquired resistance?

Spontaneous chromosomal mutation; Transfer of genetic material - transformation, transduction, or conjugation

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What are common sources of resistance genes?

Plasmids and transposons

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Name the resistance mechanism: Especially for Beta lactams and Aminoglycosides

Enzyme Degradation

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Name the resistance mechanism: Every class of antibiotics has this type of resistance

Altered target

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Name the resistance mechanism: Especially important for Gram negatives!

Porin loss or alteration
Efflux mechanism

Decreased Uptake – membrane permeability

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All cleave the Beta lactam ring

- Likely emerged before human use of beta lactam antibiotics

β-lactamases

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What Beta- lactamase does this combo inhibit? Augmentin (=Amoxicillin + Clavulanate)

Clavulanic acid

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What Beta- lactamase does this combo inhibit? Ampicillin-Sulbactam

Sulbactam

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What Beta- lactamase does this combo inhibit? Timentin (=Ticarcillin + Clavulanate)

Clavulanic acid

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What Beta- lactamase does this combo inhibit? Piperacillin Tazobactam “Pip Taz”

Tazobactam

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What’s the goal/purpose of an AST?

Test for acquired resistance (organism based) to therapeutic agents
Establish antimicrobial susceptibility profiles

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Why standardize AST?

Optimize bacterial growth conditions, Optimize conditions for maintaining antimicrobial integrity, Maintain reproducibility and consistency

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Why optimize bacterial growth conditions for AST?

To ensure that the inhibition of growth can be attributed to the antimicrobial agent.

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Why optimize bacterial growth conditions for AST?

To ensure you are attributing the failure to inhibit bacterial growth to organism-associated resistance.

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What are the standardizing components for growth medium of AST?

pH, cation concentration, blood/serum supplements, thymidine content

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What are the standardizing components for incubation of AST?

Atmosphere, temperature, duration

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What are the other two standardizing components for AST (besides growth medium and incubation requirements)?

Inoculum size and antimicrobial concentration

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Do these methods directly detect a resistance mechanism or measure antibiotic activity?

Conventional - broth dilution, agar dilution, disk diffusion
Commercial systems
Special screens & indicator tests

Directly measure antibiotic activity

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Do these methods directly detect a resistance mechanism or measure antibiotic activity? - Beta-lactamase test - PCR testing for resistance genes

Directly detect a resistance mechanism

What is the turbidity standard for AST?

0.5 McFarland turbidity standard (1.5 x 10^8 colony-forming units (CFUs) per milliliter)

How is standard turbidity achieved?

"Eyeballing it" in comparison to McFarland standard or using a turbidity meter

What is a broth dilution used for in AST?

Challenges the organism of interest with an antimicrobial agent in a liquid environment.

What are the two types of broth dilutions?

- Microdilution—Total volume is 0.05 to 0.1 mL (microtiter tray) - Macrodilution—Total volume is 1 mL or greater (tubes)

How are broth dilution tests reported?

Reported in µg/mL

What kind of dilution is commonly used for broth dilution methods?

A series of doubling dilutions

Which is the preferred method of broth dilution?

Microdilution

What are some of the reasons microdilution is preferred?

Macrodilution is too labor intensive; Microdilution is commercially prepared, automated or semi-automated, and you can run multiple drugs at once

The well with the lowest concentration of antimicrobial agent that inhibits growth

Minimum Inhibitory Concentration (“MIC”)

How do we grade organisms as a result of MICs?

Susceptible, Intermediate, Resistant, Susceptible/Dose-dependent

What is a breakpoint? Who establishes them?

Specific concentrations that define the different categories of the MIC; CLSI

What causes skipped wells?

Contamination

From an agar dilution how are MICs determined?

The MIC is the lowest concentration that inhibits visible growth.

Name the AST method described? Antibiotic disks of known concentrations are placed on the surface of an agar plate that has been seeded with a lawn of bacteria (0.5 McFarland turbidity standard).

Disk Diffusion – Kirby Bauer

What is the standard agar a Kirby Bauer test?

Mueller-Hinton agar

Why do we use Mueller-Hinton agar for a Kirby Bauer test?

- Thymidine, thymine content - Can inhibit sulfonamides (trimethoprim, SXT) - Agar depth

What is the appropriate agar depth for a Kirby Bauer test and how will results be skewed if it is too shallow or too deep?

4 mm; shallow: false SS; deep: false R

Why is cation concentration important for a Kirby Bauer test?

Ca2+ and Mg2+ concentration affects AST results for certain drug/bug combinations

What's the standard pH for AST and why is important that we maintain it?

7.2-7.4 in the test medium; A decrease (or increase) in pH of the agar surface affects activity of certain antibiotics

What's the standard incubation atmosphere for AST and why is important that we maintain it?

Ambient air, not CO2 because CO2 decreases pH of the agar and in turn may affect antibiotic activity

What is the standard temp for AST?

35-37C

What's the inoculum standard for AST?

18-24h growth, non-selective media, 0.5 McFarland, in saline

What do we use for QC for AST? How often to we perform it?

ATCC strains; performed with each use and validated weekly

What type of commercial AST is described? Commercially available panels contain dry lyophilized agents.

Broth microdilution methods

What type of commercial AST is described? Instruments apply an antimicrobial agent to the agar.

Agar dilution derivations

What type of commercial AST is described? E-test—Strips containing an antimicrobial agent generate MIC data.

Diffusion in agar derivations

What type of commercial AST is described? BIOMIC—Disk diffusion is digitally analyzed.

Diffusion in agar derivations

What type of commercial AST is described? VITEK system MicroScan WalkAway system Phoenix system

Automated test systems

What antibiotic discs do we use for supplemental agar or disc screens?

Oxacillin, Vancomycin, Cefoxitin

What is the D test used for?

Inducible Clindamycin Resistance

How does the D test ID Clindamycin resistance?

It IDs R resulting from efflux or MLS Beta methylase (coded by ERM genes)

What does a D shaped zone indicate in the D test?

Inducible Clindamycin resistance

What organisms might you perform a D test on?

S. aureus, Coagulase Negative Staphylococci, S.pneumoniae and Beta hemolytic strep

This organism has intrinsic resistance to low levels of aminoglycosides, β-lactam agents, and glycopeptides (vancomycin)

Enterococci

For what type of Enterococcus infection is combination therapy recomeneded?

Serious invasive infections

What two drug classes are uses together for HLAR? What is their target?

β-lactam agent (cell wall agent) + aminoglycoside (protein synthesis)

What agar do we use for HLAR?

Synergy quad agar

What is the predictor antimicrobial for Staph?

Oxacillin=R (Predicts resistance to all Beta lactam drugs)

What is the predictor antimicrobial for Enterococci that predicts resistance to all Pen derivatives?

Ampicillin = R (Predicts resistance to all Pen derivatives)

What is the predictor antimicrobial for Enterococci that predicts no synergy with aminoglycosides (meaning combo therapy not useful)?

HLAR +

Which drug groups do Beta lactamases hydrolyze? Which two groups does that leave?

Pens, Mononbactams and Cephalosporins; Carbapenems and Cephamycins

A chromogenic cephalosporin (Cefinase disk) is used to detect beta-lactamase production (results available within 1 to 60 minutes)

β-lactamase test

What organisms is the Beta-lactamase useful for detecting resistance in?

Neisseria gonorrhoeae Haemophilus influenzae Staphylococci B. fragilis

What is the Beta lactamase gene that can be found using PCR?

blaZ

How do you treat Staph differently for a Beta lactamase test?

For Staphylococci the β-lactamase needs to be induced by exposure to a β-lactam (grow w/ Cefoxitin disc)

- Useful for thoroughly characterizing the resistance of bacterial collections. - Important for investigating equivocal results.

Molecular methods

Downfalls of molecular testing for resistance genes

Impracticality, Use of specific probes could cause divergent genes to be missed, presence of gene doesn't mean resistance, genes may be silent and incapable of expressing resistance

What are these genes indicative of? vanA, vanB

VRE

What is the mecA gene indicative of?

MRSA

What is the bla^kpc gene indicative of?

Carbapenemases

What is the rpoB gene indicative of?

Rifampin resistance in mycobacteria

What is the principle of screening agars and what are they used for?

Soluble colorless molecules (chromogens = substrate + chromophore) are cleaved by the organism's enzyme and the chromophore is released; In this unconjugated form the chromophore exhibits a distinctive color. Screening agars are used to screen for common resistance

What are three components that make up effective AST?

Relevance, Accuracy, and Communication

Why would you NOT do AST?

Empiric therapy is sufficient for treating infection

Why would you do AST?

May help narrow the antimicrobial therapy treatment of infection, to select an adequate dose of antibiotics for treatment and to establish antimicrobial susceptibility profiles of new antibiotics

A summary report of antimicrobial susceptibility testing data from patients, which can provide valuable information for healthcare and for public health

Antibiogram

The effort to measure and improve how antibiotics are prescribed by clinicians and used by patients. Improving antibiotic prescribing and use is critical to effectively treat infections, protect patients from harms caused by unnecessary antibiotic use, and combat antibiotic resistance.

Antibiotic stewardship

What are the components of the data review process?

ID of the organism must be known, results should be analyzed and reported as early in the day as possible, two of more tiers of data are used, the review process must be flexible and updated.

For reviewing results, what are the components of resolution?

Review data for possible clerical errors, confirm that you have tested the correct isolate, confirm purity (no contamination), re-examine the test panel or plate for reading errors, use a second method to confirm resistance

What is an example of an infection that would only require empirical therapy? What would it be treated with?

Penicillin for a strep infection

What are examples of bacterial infections that may need AST?

Staph, S. pneumo, Enterococcus, Enterobacterales, Ps. aeruginosa, and Acinetobacter

What is an example of a non-bacterial infection that may indicate AST?

Yeast and some mycobacteria spp.

What are three organisms that may occasionally need AST?

H. flu, M. cat, and anaerobes

What is the most broad spectrum of all the antimicrobials?

Carbapenems - Imipenem, Meropenem, and Doripenem

``` Which class of antibiotics may be used to treat MRSA and severe C diff? ```

Glycopeptides - Vancomycin

What class of antibiotics are known to cause nephrotoxicity and ototoxicity? What is unique about the way these drugs are administered?

Aminoglycosides; they are IV only, not given orally

What class of antibiotics are often combined w/ cell wall agent for synergy?

Aminoglycosides

What class of antibiotics is known to cause bone marrow toxicity?

Chloramphenicol

What class of broad spectrum antibiotics is known to cause permanent discoloration of teeth in children <8 yr and is not for pregnant or lactating women

Tetracyclines

What class of antibiotics is only used for treating gram negative infections, specifically for serious Pseudo , Acinetobacter or MDRO infections?

Polymyxins

What class of antibiotics may be neurotoxic and/or nephrotoxic?

Polymyxins

Which class of antibiotics is limited to use for UTIs (except for infections cause by pseudomonas or proteus)?

Nitrofurantoins

What are the three broad spectrum classes of antibiotics?

Fluoroquinolones, Tetracyclines and Macrolides

Which class of antibiotics is used for Staph, S.pneumo, & strep infection in pen-allergic patients and anaerobic infections?

Lincosamides

AST Exam Flashcards

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