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Medical Microbiology lectures > L.13 AST (Antimicrobial susceptibility testing) > Flashcards

L.13 AST (Antimicrobial susceptibility testing) Flashcards

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1
Q

What is one of the core responsibilities of a diagnostic microbiology lab?

A

Antimicrobial Sensitivity Testing (AST)

AST serves several key functions including isolation and identification of pathogens and assessment of antimicrobial effectiveness.

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2
Q

What are the key functions of Antimicrobial Sensitivity Testing (AST)?

A
  • Guiding Effective Antimicrobial Therapy
  • Monitoring Antimicrobial Resistance
  • Preliminary Epidemiological Typing

These functions include selecting appropriate antibiotics, identifying resistant strains, and supporting infection control measures.

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3
Q

Why is Antimicrobial Sensitivity Testing (AST) critical?

A

To predict which antibiotics are likely to be effective for a given infection

AST helps clinicians make informed decisions about antibiotic treatments.

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4
Q

What does AST help clinicians do?

A

Select the most appropriate antibiotic

AST tailors treatment to the individual patient’s infection.

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5
Q

What does monitoring antimicrobial resistance involve?

A

Identifying resistant strains and tracking resistance trends

This can be done on local, national, and global levels.

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6
Q

What can similar susceptibility profiles among isolates indicate?

A
  • Potential cross-infection or patient-to-patient transmission
  • A cluster or outbreak in a hospital or community setting

This supports infection prevention and control measures.

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7
Q

What does the ‘S’ category in AST results indicate?

A

Susceptible, Standard Dosing

High likelihood of success with standard dosage.

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8
Q

What does the ‘I’ category in AST results indicate?

A

Susceptible, Increased Exposure

Likely to be effective if exposure is increased.

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9
Q

What does the ‘R’ category in AST results indicate?

A

Resistant

High risk of therapeutic failure even with increased exposure.

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10
Q

What are the two main approaches used to assess bacterial susceptibility?

A

Qualitative Method (Disk Diffusion) and Quantitative Methods (Minimum Inhibitory Concentration)

Disk Diffusion is also known as the Kirby-Bauer method.

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11
Q

How does the Disk Diffusion method work?

A

Bacteria are spread on an agar plate, and paper disks with antibiotics are placed on it. After incubation, zones of inhibition are measured.

Zones of inhibition indicate areas where bacterial growth is prevented by the antibiotic.

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12
Q

What does the zone diameter in the Disk Diffusion method indicate?

A

It is compared to standardized charts to report as S (Susceptible), I (Intermediate), or R (Resistant).

The zone diameter is measured in millimeters.

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13
Q

List the advantages of the Disk Diffusion method.

A
  • Simple
  • Cost-effective
  • Widely used

These advantages make it a popular choice in laboratories.

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14
Q

What is the Minimum Inhibitory Concentration (MIC)?

A

The lowest concentration of an antibiotic that visibly inhibits bacterial growth.

MIC testing can be performed using various formats.

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15
Q

What are the testing formats for MIC?

A
  • Broth microdilution
  • Agar dilution
  • Gradient diffusion strips (e.g., Etest)
  • Automated systems (e.g., Vitek, Phoenix, MicroScan)

Each format has its specific applications and advantages.

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16
Q

How are MIC values interpreted?

A

MIC values are correlated with standardized breakpoints (EUCAST or CLSI) to determine S, I, or R.

This correlation helps in clinical decision-making regarding antibiotic therapy.

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17
Q

What are Clinical Breakpoints?

A

Threshold values used to interpret AST results, defining if a microorganism is S (Susceptible), I (Susceptible, Increased exposure), or R (Resistant).

Breakpoints are specific to the pathogen, antibiotic, infection site, and AST method.

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18
Q

How is the breakpoint expressed in the Disk Diffusion method?

A

As zone diameter in millimeters (mm).

A larger zone indicates higher susceptibility.

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19
Q

What is the breakpoint for E. coli with Ampicillin in Disk Diffusion?

A

14 mm

A zone diameter ≥14 mm indicates susceptibility, while <14 mm indicates resistance.

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20
Q

How is the breakpoint expressed in the Minimum Inhibitory Concentration (MIC) method?

A

In mg/L (or µg/mL).

This measurement helps determine the effectiveness of antibiotic treatment.

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21
Q

What is the breakpoint for E. coli with Ampicillin in MIC?

A

8 mg/L

An MIC ≤8 mg/L indicates susceptibility, while >8 mg/L indicates resistance.

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22
Q

What is the relationship between zone diameter and MIC?

A

Zone diameter is inversely related to MIC: Bigger zone → Smaller MIC → More susceptible; Smaller zone → Higher MIC → More resistant.

This relationship helps in assessing the effectiveness of antibiotics.

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23
Q

Why is standardization essential for AST results?

A

Accurate, reproducible AST results depend on rigorous standardization of testing conditions across labs.

This ensures that results are comparable and reliable across different settings.

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24
Q

What are the key components to standardize in AST?

A
  • Culture Media
  • Inoculum Standardization
  • Incubation Conditions

These components are crucial for achieving consistent and accurate results.

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25
What is the requirement for culture media in AST?
Must support optimal bacterial growth without interfering with antibiotic activity. Use predefined, quality-controlled media like Mueller-Hinton agar. ## Footnote Mueller-Hinton agar is commonly used for antibiotic susceptibility testing.
26
What is inoculum standardization in AST?
Inoculum size (bacterial concentration) is a major source of variability, measured by: * McFarland Standard (typically 0.5 McF = ~1.5 × 10⁸ CFU/mL) * Densitometer for more accurate measurement ## Footnote The McFarland standard is used to ensure consistent bacterial concentrations in tests.
27
What are the standard incubation conditions for AST?
* Temperature: 35°C (±1°C) * Time: 16–18 hours * Atmosphere: Ambient air unless otherwise specified ## Footnote These conditions are standard for most non-fastidious bacteria.
28
What is the purpose of established international guidelines in AST?
To ensure consistency between labs, countries, and studies. ## Footnote This helps in maintaining the integrity and reliability of AST results globally.
29
What are the most widely used AST guidelines?
* EUCAST * CLSI ## Footnote EUCAST is primarily used in Europe, while CLSI is used in the United States and other regions.
30
What organizations provide detailed protocols for AST procedures?
EUCAST and CLSI provide: * Detailed protocols for AST procedures * Breakpoint tables for each pathogen-antibiotic pair * Interpretative criteria for assigning S, I, or R * Updates annually to reflect emerging resistance ## Footnote These protocols help standardize the interpretation of AST results.
31
True or False: England uses the same AST guidelines as the EU.
False ## Footnote England uses its own guidance (e.g., UK Standards for Microbiology Investigations), but EUCAST is dominant across Europe.
32
What are control organisms used for in AST?
To ensure accuracy and reliability in AST ## Footnote Control organisms are quality control strains tested alongside clinical isolates to provide a standard for comparison.
33
Which control strain is used for Staphylococcus spp. according to EUCAST?
Staphylococcus aureus ATCC 29213 ## Footnote This strain validates that the testing conditions are optimal.
34
Which control strain is used for Enterococcus spp. according to EUCAST?
Enterococcus faecalis ATCC 29212 ## Footnote This strain also helps in validating testing conditions.
35
Why are appropriate culture media critical for AST?
They ensure consistent and reproducible AST results ## Footnote The choice of media can affect the growth of organisms and the accuracy of results.
36
What is the standard medium for non-fastidious organisms in AST?
Mueller-Hinton (MH) agar ## Footnote It is used for organisms such as Enterobacterales, Pseudomonas spp., and Staphylococcus spp.
37
What two supplements can be added to MH agar for fastidious organisms?
5% defibrinated horse blood and 20 mg/L NAD ## Footnote These supplements are used for organisms like Streptococcus pneumoniae and Haemophilus influenzae.
38
What is the importance of inoculum preparation in AST?
Accurate inoculum density is vital for reproducibility ## Footnote Over- and under-inoculation can lead to incorrect results.
39
What is the McFarland standard used for in inoculum preparation?
0.5 McFarland standard corresponds to ~1.5 × 10⁸ CFU/mL ## Footnote This standard ensures that the inoculum density is appropriate for testing.
40
What is the time frame for inoculating agar after preparing the suspension?
Within 15 minutes ## Footnote Timeliness is crucial to maintain the integrity of the inoculum.
41
What is the method for streaking the agar plate?
Streak the entire surface of the agar plate: * First horizontally * Rotate plate 60° and streak again * Repeat a third time after another 60° rotation ## Footnote This method ensures even distribution of the inoculum.
42
What is the maximum number of discs that can be applied per 100 mm agar plate?
No more than 6 discs ## Footnote This limit helps to avoid overlapping zones which can affect result interpretation.
43
What is the standard incubation time and temperature for plates in AST?
16–20 hours at 35 ±1°C ## Footnote Prompt incubation after disc application is essential for accurate results.
44
What is the incubation condition for fastidious organisms?
4–6% CO₂ atmosphere ## Footnote This condition is necessary for organisms like S. pneumoniae and N. gonorrhoeae.
45
What should be inspected on the agar after incubation?
A confluent lawn of growth ## Footnote This indicates that conditions were appropriate for bacterial growth.
46
How should the zone of inhibition be measured?
Measure the diameter to the nearest millimetre ## Footnote Read the edge where complete inhibition ends, not where slight haze appears.
47
What can reduce variability in measuring the zone of inhibition?
Automated zone readers ## Footnote They can integrate with Laboratory Information Management Systems (LIMS) for improved efficiency.
48
What is the purpose of Control Strain Validation in AST?
Ensure that zone diameters of ATCC control strains fall within EUCAST-recommended reference ranges ## Footnote Only if controls are valid can test strain results be released.
49
What are the categories used in Test Organism Interpretation for AST?
* S (Susceptible) * I (Susceptible, Increased exposure) * R (Resistant) ## Footnote These categories indicate the likelihood of treatment success based on measured zone diameters compared to EUCAST clinical breakpoints.
50
What are the objectives of a Quality Control (QC) Program in AST?
* Ensure accuracy and precision of AST procedures * Monitor reagent performance * Evaluate the technical competence of lab personnel ## Footnote Ongoing QC practices include including control strains daily when AST is performed.
51
What should be reviewed in ongoing QC practices regarding control strains?
The last 20 consecutive results of control strains ## Footnote Look for trends, e.g., consistently smaller/larger zones. If 2 or more out of 20 are outside the acceptable range, an investigation should be initiated.
52
What does Minimum Inhibitory Concentration (MIC) refer to?
The lowest concentration of an antimicrobial agent that inhibits visible growth of a microorganism after overnight incubation ## Footnote MIC is a critical measurement in antimicrobial susceptibility testing.
53
Why was disc diffusion preferred historically over MIC determination?
MIC determination was labor-intensive ## Footnote Disc diffusion was simpler and quicker for routine susceptibility testing.
54
In what cases is MIC testing essential?
* Serious or life-threatening infections (e.g. sepsis, endocarditis) * Slow-growing organisms * Fastidious organisms * When precise antimicrobial dosing is needed ## Footnote Modern laboratories now routinely perform MIC testing due to advancements in automation.
55
True or False: Modern laboratories have reduced reliance on disc diffusion due to automation.
True ## Footnote Automation has made MIC testing more accessible and less labor-intensive.
56
Fill in the blank: The category 'I' in Test Organism Interpretation stands for _______.
Susceptible, Increased exposure ## Footnote This indicates that success is likely with dose optimization or site concentration.
57
What should initiate an investigation in Quality Control practices?
If 2 or more out of 20 control strain results are outside the acceptable range ## Footnote This helps maintain the integrity of the testing process.
58
What does MIC stand for?
Minimum Inhibitory Concentration
59
What is the purpose of MIC testing?
To expose a microorganism to a series of twofold dilutions of an antimicrobial agent
60
List the main MIC testing methods.
* Agar Dilution * Broth Dilution (Macro & Micro) * Automated AST (e.g., Vitek) * Gradient Diffusion Method (E-test)
61
What is the principle of Agar Dilution MIC?
Incorporates twofold antimicrobial dilutions into separate agar plates with spot-inoculated test and control organisms
62
Which agar is commonly used for Agar Dilution MIC?
Mueller-Hinton agar (± supplements for fastidious organisms)
63
What is the recommended inoculum volume for Agar Dilution MIC?
1–2 μL (spot inoculum) of 10⁴ CFU/mL suspension
64
How many organisms can be spotted per plate in Agar Dilution MIC?
Up to 27 organisms
65
What temperature and duration are required for incubation in Agar Dilution MIC?
Overnight at 35°C ±1°C
66
How is the MIC determined in Agar Dilution MIC?
Lowest concentration showing no visible growth
67
What should be compared to determine the clinical relevance of the MIC?
MIC of test strains to clinical breakpoints for the relevant organism (EUCAST/CLSI)
68
What are the advantages of the Agar Dilution MIC method?
* Allows simultaneous testing of many isolates * ATCC and test strains on the same plate
69
What are the limitations of the Agar Dilution MIC method?
* Time-consuming and labour-intensive * Antimicrobials may degrade quickly (shelf life ~1 week) * Requires pure-grade antimicrobials from pharmaceutical suppliers
70
What is the principle of Broth Dilution MIC?
Twofold antimicrobial dilutions in Mueller-Hinton broth inoculated with a standardised bacterial suspension
71
What are the two types of Broth Dilution MIC?
* Macrobroth: Larger volumes (1–2 mL) * Microbroth: Smaller volumes (50–100 µL)
72
What is the most common technique used in Microbroth Dilution?
Performed in 96-well microtitre trays
73
What are the advantages of the Microbroth Dilution method?
* Compatible with automation (e.g. Microscan, Sensititre) * Efficient for high-throughput testing
74
What are the limitations of the Microbroth Dilution method?
* Difficult to detect contamination * Not ideal for fastidious organisms (may not grow well in small volumes)
75
What is the principle of Automated AST Systems?
Automated systems use micro-well dilution format, testing 3–4 antibiotic concentrations targeting breakpoints for S, I, R ## Footnote Automated systems like Vitek integrate organism ID and MIC determination.
76
What types of organisms are targeted by AST panels in Automated AST Systems?
* Enterobacterales * Pseudomonas aeruginosa * Staphylococcus spp. * Enterococcus spp. ## Footnote These panels are specifically designed for these groups.
77
What is the incubation time for Automated AST Systems?
4–16 hours ## Footnote This short incubation time helps in faster result generation.
78
What are the advantages of Automated AST Systems?
* Good correlation with manual MIC * High reproducibility * Reduced labor & faster turnaround * Interfaces with LIMS * Automated result interpretation and resistance trend detection ## Footnote These advantages make automated systems efficient in clinical settings.
79
What are the limitations of Automated AST Systems?
* Not suitable for slow-growing or fastidious organisms * Limited dilution range tested → may not provide exact MIC value ## Footnote These limitations can affect the effectiveness of the testing in certain situations.
80
What is the principle of the Gradient Diffusion Method (E-test)?
Combines MIC dilution and disc diffusion principles using a plastic strip impregnated with an antimicrobial gradient ## Footnote The drug diffuses into agar, forming an elliptical zone of inhibition.
81
What type of agar is used in the Gradient Diffusion Method?
Mueller-Hinton agar (± supplements) ## Footnote This media is standard for antimicrobial susceptibility testing.
82
What is the inoculum standard for the Gradient Diffusion Method?
0.5 McFarland standard, spread evenly over plate ## Footnote This standard ensures consistent inoculum density for reliable results.
83
What are the advantages of the Gradient Diffusion Method?
* Simple, easy-to-use * Ideal for individual isolate MIC testing * Suitable for fastidious organisms * Useful when automation isn't available ## Footnote These features highlight its practicality in various laboratory settings.
84
What are the limitations of the Gradient Diffusion Method?
* Expensive (~€3 per strip) * Must test control strain separately * Not ideal for large-scale testing ## Footnote These limitations can restrict its use in high-throughput environments.
85
What is the first step in MIC interpretation?
Validate control strains (ATCC) ## Footnote MICs must fall within published reference ranges to ensure accuracy.
86
What is the second step in MIC interpretation?
Interpret test strain MICs by comparing to clinical breakpoints (EUCAST or CLSI) ## Footnote This comparison is crucial for determining susceptibility.
87
How are MIC results categorized?
* S – Susceptible * I – Susceptible with increased exposure * R – Resistant ## Footnote This categorization helps guide treatment decisions.
88
What condition must be met before releasing MIC results?
Control MICs must be valid ## Footnote Valid control MICs ensure the reliability of test results.

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