Module 7: Antimicrobial Susceptibility Tests Flashcards
When might antimicrobial susceptibility not be performed?
When the bacteria is known to be universally susceptible to certain antibiotics
ex: S. pyogenes with Pen G, S. pneumoniae with Pen G, anaerobes
What are 3 conventional methods for performing susceptibility testing?
Broth, agar, and disc diffusion
How is a standard bacterial inoculum achieved for susceptibility testing?
- 3-10 colonies from a primary plate used
- Colonies in log growth phase (after 18-24 hour incubation)
- Suspension made up to 0.5 McFarland standard
Why are bacteria in the log growth phase desired for susceptibility testing?
They are actively metabolizing and most sensitive to antibiotics
What are the direct and indirect methods used in inoculated broth for susceptibility?
Indirect - colonies emulsified and then incubated until turbid
Direct - colonies emulsified until they reach 0.5 McFarland
What growth medium has been standardized for susceptibility testing?
Mueller Hinton agar or broth
What does the Mueller Hinton broth/agar contain?
Beef infusion Acid hydrolysis of casein Starch Water (Agar for MH agar) pH = 7.2-7.4
What cautions must be observed in the test medium?
- Calcium and magnesium concentration close to that of the body
- Check for thymidine with QC organisms
What might improper calcium and magnesium level in the test medium do to the susceptibility results?
Affects aminoglycoside movement
Too much mag/cal = prevent movement = false resistance
Too little mag/cal = more movement = false sensitivity
What would happen if too much thymidine was in the test medium?
Interferes with sulfonamides and trimethoprim
Bacteria can use thymidine to bypass PABA to folic acid pathway
Too much = false resistance
What are two methods of broth dilution susceptibility testing?
Macro and micro
What happens in the macro broth dilution susceptibility test?
Antibiotic diluted in serious of doubling dilutions
Inoculum added to each tube
Incubated overnight and inspected for visible growth/turbidity
Lowest inhibitory concentration determind
Define minimum inhibitory concentration
Lowest concentration of antimicrobial in mg/L that prevents in vitro growth of the organism
Define minimum bactericidal concentration
Lowest concentration of antimicrobial in mg/L that results in more than 99.9% killing in vitro
How is MBC determined?
Taking all no growth MIC tubes and planting them to a plate then incubated overnight.
The plate with 0.01% growth to no growth with the smallest dilution is the MBC
What are the two controls included on a microdilution susceptibility test? (Many welled plate)
Sterility well - does not get inoculated with bacteria, should have no growth
Growth control - does not have antimicrobials, should have growth
What are the advantages of automated susceptibility systems?
Quick results - usually same day reporting
Accurate
Less technologist time
Why are purity plates required for automated susceptibility testing methods?
To ensure that the bacteria sampled is only of one type
What is involved in agar dilution susceptibility testing?
Antimicrobials are diluted in the agar itself, standard inoculum is used, plates are incubated, MIC is read the same was as in a broth susceptibility test
What adaptations can be made to make agar dilution sensitivity testing feasible routinely?
Limit the number of plates used by only using dilutions within the antimicrobials theraputic range
Single plate representing the break point for each antimicrobial, growth = resistant
What are advantages of agar dilution susceptibility testing?
Cheap if plates are readily available
Customizable test for different microbials and urinary concentrations
Adaptive testing for fastidious organisms (special nutrients required)
What manual test is most often used for antimicrobial sensitivity?
Disc diffusion on Kirby Bauer plates
What is the principle of Kirby Baur disc diffusion sensitivity testing?
Antibiotic discs are placed on agar after streaking with inoculum and the plate is incubated
Antimicrobials diffuse out of the disc and inhibit bacterial growth
A zone of inhibition is made (no growth) and measured to determine sensitivity
What factors affect antibiotic diffusion rate in KB disc diffusion?
Agar depth (4-6mm) - thin = greater diffusion, thick = less diffusion Composition - stiff = less diffusion Moisture and pH
What does “critical point” in KB disc diffusion mean?
The lowest concentration of antibiotics that inhibits growth (where the growth stops)
What is a regression graph?
Shows relation between MIC and disc zone of inhibition measurements
What happens to a KB disc diffusion test if the standard inoculum is not met?
Standard = 0.5 McFarland Higher = smaller zone of inhibition = resistance Lower = bigger zone of inhibition = sensitivity
What may happen on KB disc diffusion when using sulfonamides or trimethoprim discs?
A fine veil of growth within the zone of inhibition, even if the organism is susceptible (will continue to grow until folic acid is used up)
What may happen on KB disc diffusion when using penicillin discs?
Sharp irregular zones around beta-lactam antibiotics
Beta-lactamase is produced by the bacteria
INTERPRET AS RESISTANT
What does moderately susceptible mean?
Implies that bacteria can be treated by attainable concentration of the antimicrobial, provided higher dosages are used, or in a body site (urine) where drugs are concentrated
What are some limitations of KB disc diffusion sensitivity?
- Bacteria must form colonies overnight, now slow growers
- Bacteria must be able to grow on the Mueller Hinton agar without enrichment (not fastidious)
- Bacteria must be able to grow in O2
When and how is QC done for KBDD?
Set by the lab, when new discs/panels are received, with each susceptibility test (or possibly weekly)
Done with S. aureus, E. coli, and P. aeruginosa
MIC values are already established
How does antimicrobial discs be stored?
At -20 or lower in moisture free environment
Working stock at 4 (fridge) with desiccant
How are modifications for KBDD done for MRSA?
Add NaCl to media and broth to enhance growth
Incubate to 30-35, grows better at cooler temperature
“Trailing” may be seen at edge of ZOI
What screening is done for MRSA?
Oxacillin screen test (agar dilution)
MH agar with 6ug/mL oxacillin and 4% NaCl
Spot inoculated to the plate
Growth = resistance
What screening is done for penicillin resistant S. pneumoniae?
MH enriched with blood 1ug oxacillin disc Incubate in CO2 ZOI >/= 20mm = sensitive Smaller = resistant
How is susceptibility done for Hemophilus? Why?
Hemophilus test medium used with bovine hematin, yeast extract, NAD, and thymidine phosphorylase
Done because Hemophilus is fastidious and will not grow ever on MH or in the broth
How is susceptibility done for N. gonorrhoeae
Agar dilution in CO2 with necessary agar nutrients
or
Modified disc diffusion with GC enriched agar base incubated in CO2
How are E-tests used?
Same as disc diffusion
Agar with 0.5 McFarland inoculum, put on strip, incubate, read for MIC from elliptical zone of inhibition
What are the advantages and disadvantages of the E-test?
Ad: can be used with anaerobes and fastidious bacteria that regular KBDD cannot
Dis: expensive, may be more difficult to read
The main objective of susceptibility testing is to
a. inform the physician of the one best antimicrobial for treatment
b. provide the physician with a list of effective antimicrobials
c. provide statistic for infection control purposes
d. create more positions in the laboratory
b. provide the physician with a list of effective antimicrobials
Some organisms do not have routine susceptibility tests performed because
a. antimicrobials are not used for treatment
b. standardized test methods are not available
c. universally susceptible to a suitable antimicrobial
d. both b and c are correct
d. standardized test methods are not available and universally susceptible to a suitable antimicrobial
The pH of Mueller Hinton agar used for susceptibility testing should be
a. 7.2-7.4 before sterilization
b. 7.2-7.4 when the medium is ready to use
c. 6.8-7.6 for the finished product
d. slightly acidic
b. 7.2-7.4 when the medium is ready to use
The concentration of divalent cations in susceptibility test medium effects results for
a. aminoglycosides
b. beta-lactams
c. quinolones
d. cephalosporins
a. aminoglycosides
Antibiotic concentration of 2ug/mL expressed in SI units is
a. 200mg/mL
b. 2mg/mL
c. 2mg/L
d. 200mg/L
c. 2mg/L
An antibiotic solution containing 500 untis/mL is diluted in doubling dilutions. This would give the following concentrations
a. 500, 400, 300, 200
b. 500, 100, 50, 25
c. 500, 250, 125, 62.5
d. 500, 50, 5, 1
c. 500, 250, 125, 62.5
Minimum inhibitory concentration is defined as
a. maximum amount of antimicrobial inhibiting growth in vitro
b. minimum amount of antimicrobial inhibiting growth in vivo
c. maximum amount of antimicrobial killing bacteria in vitro
d. minimum amount of antimicrobial inhibiting growth in vitro
d. minimum amount of antimicrobial inhibiting growth in vitro
Escherichia coli tested against ampicillin gave an MIC of 0.2mg/L. If average blood levels following administration of ampicillin are 2.0mg/L, this result would be interpreted as
a. susceptible
b. resistant
c. intermediate
d. moderately susceptible
a. susceptible
Escherichia coli tested against ampicillin gave an MIC of 0.2mg/L. If average blood levels following administration of ampicillin are 2.0mg/L, this result, if isolated from a urinary infection, would be interpreted as
a. susceptible
b. resistant
c. intermediate
d. moderately susceptible
a. susceptible
Minimum bactericidal concentration determinations are
a. performed on all toxic antimicrobials
b. only valid for bactericidal antimicrobials
c. used to determine if an antimicrobial is penetrating a specific tissue
d. available the same day as the MIC
b. only valid on bactericidal antimicrobials
The concentration of antimicrobial at the edge of the zone of inhibition in the Kirby Baur test corresponds to
a. the MBC
b. the MIC
c. the break point between susceptible and resistant
d. both b and c
b. the MIC
The following observation may indicate a mixed culture in the Kirby Baur susceptibility test
a. scalloped edges of zones of inhibition
b. sharp irregular zones of inhibition
c. confluent growth around zones of inhibition
d. colonies within the zones of inhibition
d. colonies within the zones of inhibition
Leaving Mueller Hinton plates at room temperature for one hour after discs have been applied will
a. not affect results
b. give false susceptible results
c. give false resistant results
b. give false susceptible results
A fine veil of growth inside the main zone of inhibition may be ignored with
a. oxacillin
b. sulfonamides
c. aminoglycosides
d. none of the above
b. sulfonamides
Irregular zones with sharp edges may be seen around the following discs
a. trimethoprim
b. aminoglycosides
c. sulfonamides
d. penicillin
d. penicillin
Which of the following often indicates a problem with the accuracy of Kirby Bauer results
a. most results are susceptible
b. most results are resistant
c. most results are intermediate
c. most results are intermediate
An antimicrobial susceptibility test result reported as intermediate means
a. the zone size could not be accurately measured
b. uncertain as to whether the antimicrobial will be effective
c. some of the bacteria showed resistance
d. the organism is susceptible to a less expensive antimicrobial
b. uncertain as to whether the antimicrobial will be effective
The quality control organism used for checking antimicrobial susceptibility for gram positive cocci is
a. Escherichia coli
b. Pseudomonas aeruginosa
c. Streptococcus pyogenes
d. Staphylococcus aureus
d. Staphylococcus aureus
The quality control results show zone sizes are too small for sulfonamides while other antimicrobials are in control. The problem may be
a. agar depth is too thin
b. thymidine level may be too high
c. divalent cations may be too low
d. inoculum density is too high
b. thymidine level may be too high
Quality control results show zone sizes are too small for ampicillin while other antimicrobials are in control. The problem may be
a. agar depth too thick
b. inoculum density too high
c. plates left out of incubator too long after discs applied
d. ampicillin discs may have lost potency
d. ampicillin discs may have lost potency
The following may have more than 1 answer:
Conventional methods of performing susceptibility tests include
a. broth dilution
b. agar dilution
c. disc diffusion
d. epsilometer test
a, b, and c
broth dilution, agar dilution, and disc diffusion
The following may have more than 1 answer:
The standardized inoculum used for susceptibility testing
a. contains 1.5x10^8 CFU/mL
b. is derived from a single colony
c. is in the log growth phase
d. matches the 1.5 McFarland standard
a and c
contains 1.5x10^8 CFU/mL and is in the low growth phase
The following may have more than 1 answer:
The concentration of thymidine in susceptibility test medium affects results for
a. aminoglycosides
b. tetracycline
c. beta-lactams
d. sulfonamides
d. sulfonamides
The following may have more than 1 answer:
In the macro dilution method of susceptibility testing
a. doubling dilutions of antimicrobials are used
b. a standardized inoculum is used
c. tests are incubated overnight
d. growth is detected by gram staining each tube.
a, b, and c
doubling dilutions of antimicrobials are used, a standardized inoculum is used, and tests are incubated overnight
The following may have more than 1 answer:
When performing a MBC determination
a. all clear tubes from the MIC are subcultured
b. the MBC will be lower than the MIC
c. the test requires 48 hours
d. two fold dilution of antimicrobial are made in MH agar
a and c
all clear tubes from the MIC are subcultured and the test requires 48 hours
The following may have more than 1 answer:
Commercial plates or trays for microdilution susceptibility tests are available
a. frozen
b. diluted in agar
c. lyophilized
d. pre-inoculated with QC bacteria
a and c
frozen and lyophilized
The following may have more than 1 answer:
The Vitek system for antimicrobial susceptibility testing
a. uses a standard inoculum
b. determines MIC values
c. provides results overnight or sooner
4. tests urinary isolates with higher concentrations of antimicrobials
a, b, c, and d
uses a standard inoculum, determines MIC values, provides results overnight or sooner, and tests urinary isolates with higher concentrations of antimicrobials
The following may have more than 1 answer:
Advantages of the Vitek system include
a. provides MBC results
b. detects mixed cultures
c. determines serum levels of antimicrobials
d. interpretation of test results
d. interpretation of test results
The following may have more than 1 answer:
The agar dilution method of susceptibility testing
a. may be adapted for fastidious bacteria
b. does not require a standardized inoculum
c. provides an MIC result
d. inoculates only one bacterial isolate per plate
a and c
may be adapted for fastidious bacteria and provides an MIC result
The following may have more than 1 answer:
The following factors affect the rate of diffusion of antimicrobials on Mueller Hinton agar
a. structure of the antimicrobial molecules
b. agar depth
c. pH of medium
d. concentration of agar
a, b, c, and d
structure of the antimicrobial molecules, agar depth, pH of medium, concentration of agar
The following may have more than 1 answer:
The following factors may cause larger zones of inhibition in disc diffusion susceptibility tests
a. 10^9 CFU/mL in the inoculum
b. thin agar plates
c. testing colonies from a sub plate
d. bacterial cells in inoculum being in the stationary growth phase
b and d
thin agar plates and bacterial cells in inoculum being in the stationary growth phase
The following may have more than 1 answer:
The procedure used to correlate zone sizes with susceptibility in disc diffusion tests
a. plots MICs and zone sizes for a large number of bacteria
b. uses antimicrobial serum levels
c. requires a regression graph for each antimicrobial
d. uses antimicrobial urine levels
a, b, and c
plots MICs and zone sizes for a large number of bacteria, uses antimicrobial serum levels, and requires a regression graph for each antimicrobial
The following may have more than 1 answer:
The following incubation conditions for Kirby Bauer susceptibility tests are acceptable
a. anaerobic
b. 3-5% carbon dioxide
c. 37 degrees
d. ambient air
d. ambient air
The following may have more than 1 answer:
Kirby Bauer susceptibility tests are suitable for
a. Staphylococcus
b. Enterococcus
c. Enterobacteriaceae
d. Hemophilus
a, b, and c
Staphylococcus, Enterococcus, and Enterobacteriaceae
The following may have more than 1 answer:
The following modifications of susceptibility tests are designated to help detect MRSA
a. preparing the inoculum by the direct method
b. increasing the concentration of divalent cations
c. incubating at 30 degrees
d. adding thymidine phosphorylase
a and c
preparing the inoculum by the direct method and incubating at 30 degrees
The following may have more than 1 answer:
Modified disc diffusion methods may be used for testing the following organisms
a. Streptococcus pneumoniae
b. Hemophilus influenzae
c. Neisseria gonorrhoeae
d. anaerobes
a, b, and c
Streptococcus pneumoniae, Hemophilus influenzae, and Neisseria gonorrhoeae
The following may have more than 1 answer:
The Epsilometer test (E-test)
a. uses a solid medium streaked with a standardized inoculum
b. uses plastic strips with concentration gradient of antimicrobials
c. provides MIC result
d. is suitable for testing fastidious bacteria
a, b, c, and d
uses a solid medium streaked with a standardized inoculum, uses plastic strips with a concentration gradient of antimicrobials, provides an MIC result, and is suitable for testing fastidious bacteria
