Module 7 Flashcards
Goal of susceptibility testing
provide a list of antimicrobials that would be effective against the organism responsible for the infection
In vitro
in the lab
In vivo
In the body
Questions Dr has to ask to determine if its a good antimicrobial to prescribe (4)
Is the antimicrobial effective at the pH found at the site of infection?
Will the antimicrobial penetrate to the site of infection?
Is it toxic to host tissue?
Is it cost effective?
3 conventional methods of performing antimicrobial susceptibility testing
broth dilution
agar dilution
disc diffusion
of colonies selected for inoculum
3-10 colonies taken from primary plate
Mixed cultures cannot be used
Growth phase of inoculum
Log growth phase when cells are most active
Indirect method
Selected colonies are inoculated to a broth and incubated for 4hrs until visually turbid (entering log phase)
Direct method
colonies in log phase are selected and emulsified in a broth to reach the turbidity standard needed
Standardizing inoculum
inoculated broth is standardized to 0.5 McFarland standard (1.5 x 10^8 CFU/mL)
Sterile saline or broth is added until it matches the standard if the turbidity it too heavy
Growth medium for susceptibility testing
Mueller Hinton (MH) broth or agar Contains: beef infusion, acid hydrolysis of casein, starch, water, agar for MH agar Final pH should be 7.2-7.4
Cations in growth medium
Concentration of Ca and Mg must be close to body tissue levels (physiological concentration)
Ca and Mg affect rate of movement of aminoglycosides into the cell
Low concentration = more antibiotic moves into cell = false susceptible results (falsely large zones)
High concentration = less antibiotic moves into cell = false resistance results (falsely small zones)
Check quality control of levels using Pseudomonas aeruginsoa with Aminoglycoside antibiotic
Thymidine concentration in growth medium
May interfere with results for sulphonamides and trimethoprim
Some bacteria use thymidine and bypass PABA to folic acid pathway that is inhibited by these antimicrobials (shows false resistance)
Media summary
Meuller Hinton broth or agar
pH 7.2-7.4
Divalent cations, Ca and Mg need to be at corrected concentrations for testing Aminoglycosides
Keep Thymidine level low for Sulphonamide and Trimethoprim testing
2 types of Broth dilution
Macro methods - manual method that may be used as a reference method
Micro methods - use in semi and fully automated methods
Macro Dilution method
Antimicrobial is diluted - antimicrobial + broth (doubling dilutions = 200, 100, 50, 25, 12.5, 6.25, 3) into each test tube.
A standardized inoculum of the organism is added to each tube
All tubes have the same total mL in them
Last tube gets no antimicrobial and serves are growth control
Tubes incubated overnight @ 35C in ambient air
Macro Dilution concentration
usually ug/mL but should be mg/L (SI units)
Number stays the same when converting the two
10ug/mL = 10mg/L
Macro Dilution math
V1C1=V2C2
MIC
Minimal inhibitory concentration is the lowest concentration of an antimicrobial in mg/L that prevents in vitro growth of the organism
Control must show growth
For an antimicrobial to be effective in vivo:
the in vivo level (determined by manufacturer/pharmacological info) should be 2-4x higher than the in vitro MIC
Urine levels are usually much higher than blood levels
MBC
Minimal Bactericidal Concentration is the lowest concentration of antimicrobial (mg/L) that results in more than 99.9% killing in vitro
Can only be determined for bactericidal antimicrobials
MIC must first be determined. For all tubes showing no growth, subculture to an agar medium with no other antimicrobials. Incubate over night and read for growth.
The MBC would never be lower than the MIC
Microdilution Broth Susceptibility tests
Based on same principles as macro method
Plastic plates with molded micro tube wells (100 or more)
Allows doubling dilutions of 10 or more antimicrobials per plate
Frozen at -20C or lyophilized at RT 4degC
Reading micro dilution plates
Incubated overnight @35C in ambient air
2 control wells to read first:
Sterility Well: S contains growth medium that is not inoculated (should be clear, no turbidity or growth)
Growth control: G contains the growth medium (no antimicrobial) and is inoculated with a probe (should show growth, turbidity or “button” of growth in well)
Vitek
automated system for antimicrobial susceptibility testing and organism identification by biochemical tests
Small microcell cassette or cards for Gram pos, gram neg and urine
Vitek 2
the standardized inoculum is drawn into the wells of the cassette in a vacuum chamber module of the instrument. Cassettes are loaded into the reader-incubation module. Each cassette is read photometrically for growth at regular intervals The results are plotted by the computer and MIC and interpretation are printed
Advantages of vitek
Same day results (sometimes even 6hrs)
Accurate, less chance of technical error
Time saving
Maldi-Tof technology for identification of bacteria directly from pt sample
Mixed cultures
Subculture the standardized inoculum to check for purity
Purity must be observed before pt results may be released
Agar Dilution susceptibility tests
Seldom used for routine testing
similar to broth dilution except the antimicrobials are diluted in agar instead of broth (still at doubling dilutions)
Plates are inoculated with standard amount of inoculum, incubated overnight and inspected for growth
MIC is determined the same way as broth dilution tests
Adaptations to make agar dilution feasible for routine use (2)
-total number of plates can be reduced to a few plates around the therapeutic range for each antimicrobial (3-4 plates)
-single plate with break points for each antimicrobial (growth = resistance, no growth= susceptibility)
Sometimes used to screen for resistance strains of specific bacteria
Steers replicator
instrument used to inoculate plates with multiple wells at once
Advantages of agar dilution testing
cheap if plates are readily available
Can customize test for different antimicrobials and for urinary concentrations
Can adapt the test for fastidious organisms
Kirby Bauer sensitivity test
Disc diffusion testing
A standard inoculum is spread over entire surface of agar plate (lawn)
Discs placed onto surface of plate
During incubation, antimicrobial diffuses into the agar (down and then out) to form a circular zone around the disc.
Colonies do not form where there is sufficient antimicrobial to inhibit growth (zone of inhibition)
4-6 hours for colonies to START growing (if in log phase)
Effects of agar on kirby bauer discs
thin agar = falsely large zone
thick agar = falsely small zone
stiff agar = falsely small zone
Surface moisture and pH also affect diffusion of some antimicrobials
If colonies are not in log phase during kirby bauer test
they will take longer to start growing
The antimicrobial would keep diffusing away from the disc and the result would be larger zones of inhibition = falsely sensitive
Critical point
the lowest concentration of antimicrobial around the disc that inhibits growth
Corresponds to the MIC in a broth dilution test
Interpretation of zone sizes
Measured with ruler across entire zone of inhibition (including width of disc)
You will NEVER have a measurement of 0mm
No zone of inhibition = width of disc in mm
Labeled as Susceptible, Resistant or Intermediate
Regression graph
zone size and MIC are determined for each organism and plotted on graph
Regression lion is drawn through points (line of best fit)
Kirby Bauer procedure
Select 4-5 colonies (no mixed cultures) from agar plate, transfer to tube of 4-5mL broth
Incubate until at 0.5 McFarland (indirect) or add more colonies/broth until it reads 0.5 McFarland (direct)
Within 15 min, lawn the MH plate
Allow to dry for 3-5min and place discs on the surface
Within 15 min, place plates into incubator at 35C
After 16-24hrs, measure zones of inhibition for each antimicrobial
Report at S, R or I
Standardization of inoculum density
must be 0.5 McFarland (1.5x10^8 CFU/mL)
More CFU/mL = smaller zones = falsely resistant
Less CFU/mL = larger zones = falsely sensitive
Meuller Hinton agar requirements
90mm plates or large 140mm plates
agar depth 4-6mm
Thin agar = larger zones of inhibition (falsely sensitive)
Thick again = smaller zones of inhibition (falsely resistant)
Plates should have been poured level
Streaking plates for Kirby Bauer testing (lawn)
cotton swab dipped in standardized inoculum is streaked across the entire surface of the plate in at least 3 directions
Non circular zones or “scallops” around disc occur if swab was too wet or plate was streaked when pressing too hard
Application of discs
must be within 15 min of swabbing
If longer than 15min, bacteria get a head start before antimicrobials gets to diffuse = smaller zones = falsely resistant
Press disc firmly onto surface
Plates must be incubated within 15 min of placing discs
Measuring zones
Some bacteria are mobile and will swarm into the zone of inhibition
Resistant colonies may grow into zone of inhibition (check purity, if pure they are resistant colonies not contamination)
Inner veil of fine growth form sulphonamide and trimethoprim antimicrobials (measure where 80% or more is inhibited)
Unusual zone of inhibition around beta-lactam antibiotics
S
means organism is susceptible (sensitive) to action of that antimicrobial and is killed by or or stops growing
R
means organism is resistant to the action of the antimicrobial and keeps growing in its presence
I
Intermediate - produces results of uncertain significance
MS
Moderately susceptible
higher dosage needed in body site to cure this infection
Limitations of Kirby Bauer testing
Can only be used for bacteria that:
form colonies overnight (fast growers)
can grow on MH agar without addition of nutrients
can grow in ambient air without increased CO2
Good for: staphylococci, enterococci, enterobacteriaceae, non-fermentative gram neg rods
QC of Kirby Bauer
each time new discs or antimicrobial panels are received
Each time susceptibility tests are performed
Staphylococcus aureus ATCC 25923
Escherichia coli ATCC 25922
Pseudomonas aeruginosa ATCC 27853
Acceptable MIC and zone sized must be established
QC must be accepted before pt results may be released
ATCC
American Type Culture Collection
Storage of antimicrobial discs
stored at -20C or colder in a moisture free environment
Working supply used on bench daily may be stored at 4C in fridge
Should be warmed to RT before use to prevent condensation when placed on the plates
Modifications to detect MRSA
Prepare standard inoculum by direct method (because MRSA are slow growing)
Addition of NaCl to MH broth and again to enhance growth
Incubate between 30-35C
Resistant strains grow better at lower temps
MRSA typically shows “tailing” at the edge of the zone of inhibition
Oxacillin screen test for MRSA
Meuller Hinton agar with 6ug/mL oxacillin and 4%NaCl.
Standard inculum using direct method on a spot on the surface of the plate
After incubation, any growth on plate is interpreted as resistance and further testing is carried out
Example of an agar dilution method
Penicillin resistant S. pneumoniae
standard inoculum prepared by direct method and streaked onto entire MH plate enriched with blood
1ug oxacillin disc applied to surface
Incubated in CO2 and zones of 20mm or more indicate susceptibility to penicillin
Smaller zones or colonies within the zone indicate possible resistance and MIC determinations are recommended
Susceptibility tests for Haemophilus
fastidious bacteria that do not grow on MH broth or agar
Standard inoculum prepared by direct method
Modified MH agar called Haemophilus Test Medium (HTM) with boxing hematin, yeast extract, NAD, thymidine phosphorylase
Susceptibility tests for N. gonorrhoeae
fastidious bacteria
Agar dilution test may be used to test medium containing necessary nutrients and incubated in CO2
Modified disc diffusion:
Inoculum by direct method
GC agar base enriched to allow growth
plates incubated in CO2
The E-Test
The Epsilometer Test
Plastic strip with predefined antimicrobial agent concentration gradient and MIC scale covering 15 dilutions
Show elliptical inhibitory zones
Expensive
Good for anaerobes and fastidious bacteria
E-test procedure
Warm strips to RT
Swab plate with bacterial suspension
Use forecast to pull test strip from cartilage and lay gently on surface of agar (one end at a time)
Push strip onto surface to remove any bubbles
Incubate late within 15min of strip application (agar side up)
Kirby bauer results vs E-test results
Kirby Bauer:
Large zone of inhibition = sensitive result
Small zone of inhibition = resistant result
E-Test:
Low MIC value = sensitive result
High MIC value = Resistant result
