Module 7

Question 1

Goal of susceptibility testing

Answer 1

provide a list of antimicrobials that would be effective against the organism responsible for the infection

Question 2

In vitro

Answer 2

in the lab

Question 3

In vivo

Answer 3

In the body

Question 4

Questions Dr has to ask to determine if its a good antimicrobial to prescribe (4)

Answer 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?

Question 5

3 conventional methods of performing antimicrobial susceptibility testing

Answer 5

broth dilution
agar dilution
disc diffusion

Question 6

of colonies selected for inoculum

Answer 6

3-10 colonies taken from primary plate

Mixed cultures cannot be used

Question 7

Growth phase of inoculum

Answer 7

Log growth phase when cells are most active

Question 8

Indirect method

Answer 8

Selected colonies are inoculated to a broth and incubated for 4hrs until visually turbid (entering log phase)

Question 9

Direct method

Answer 9

colonies in log phase are selected and emulsified in a broth to reach the turbidity standard needed

Question 10

Standardizing inoculum

Answer 10

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

Question 11

Growth medium for susceptibility testing

Answer 11

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

Question 12

Cations in growth medium

Answer 12

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

Question 13

Thymidine concentration in growth medium

Answer 13

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)

Question 14

Media summary

Answer 14

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

Question 15

2 types of Broth dilution

Answer 15

Macro methods - manual method that may be used as a reference method
Micro methods - use in semi and fully automated methods

Question 16

Macro Dilution method

Answer 16

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

Question 17

Macro Dilution concentration

Answer 17

usually ug/mL but should be mg/L (SI units)
Number stays the same when converting the two
10ug/mL = 10mg/L

Question 18

Macro Dilution math

Answer 18

V1C1=V2C2

Question 19

MIC

Answer 19

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

Question 20

For an antimicrobial to be effective in vivo:

Answer 20

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

Question 21

MBC

Answer 21

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

Question 22

Microdilution Broth Susceptibility tests

Answer 22

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

Question 23

Reading micro dilution plates

Answer 23

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)

Question 24

Vitek

Answer 24

automated system for antimicrobial susceptibility testing and organism identification by biochemical tests
Small microcell cassette or cards for Gram pos, gram neg and urine

Question 25

Vitek 2

Answer 25

``` 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 ```

Question 26

Advantages of vitek

Answer 26

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

Question 27

Mixed cultures

Answer 27

Subculture the standardized inoculum to check for purity | Purity must be observed before pt results may be released

Question 28

Agar Dilution susceptibility tests

Answer 28

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

Question 29

Adaptations to make agar dilution feasible for routine use (2)

Answer 29

-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

Question 30

Steers replicator

Answer 30

instrument used to inoculate plates with multiple wells at once

Question 31

Advantages of agar dilution testing

Answer 31

cheap if plates are readily available Can customize test for different antimicrobials and for urinary concentrations Can adapt the test for fastidious organisms

Question 32

Kirby Bauer sensitivity test

Answer 32

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)

Question 33

Effects of agar on kirby bauer discs

Answer 33

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

Question 34

If colonies are not in log phase during kirby bauer test

Answer 34

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

Question 35

Critical point

Answer 35

the lowest concentration of antimicrobial around the disc that inhibits growth Corresponds to the MIC in a broth dilution test

Question 36

Interpretation of zone sizes

Answer 36

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

Question 37

Regression graph

Answer 37

zone size and MIC are determined for each organism and plotted on graph Regression lion is drawn through points (line of best fit)

Question 38

Kirby Bauer procedure

Answer 38

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

Question 39

Standardization of inoculum density

Answer 39

must be 0.5 McFarland (1.5x10^8 CFU/mL) More CFU/mL = smaller zones = falsely resistant Less CFU/mL = larger zones = falsely sensitive

Question 40

Meuller Hinton agar requirements

Answer 40

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

Question 41

Streaking plates for Kirby Bauer testing (lawn)

Answer 41

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

Question 42

Application of discs

Answer 42

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

Question 43

Measuring zones

Answer 43

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

Question 44

S

Answer 44

means organism is susceptible (sensitive) to action of that antimicrobial and is killed by or or stops growing

Question 45

R

Answer 45

means organism is resistant to the action of the antimicrobial and keeps growing in its presence

Question 46

I

Answer 46

Intermediate - produces results of uncertain significance

Question 47

MS

Answer 47

Moderately susceptible | higher dosage needed in body site to cure this infection

Question 48

Limitations of Kirby Bauer testing

Answer 48

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

Question 49

QC of Kirby Bauer

Answer 49

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

Question 50

ATCC

Answer 50

American Type Culture Collection

Question 51

Storage of antimicrobial discs

Answer 51

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

Question 52

Modifications to detect MRSA

Answer 52

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

Question 53

Oxacillin screen test for MRSA

Answer 53

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

Question 54

Penicillin resistant S. pneumoniae

Answer 54

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

Question 55

Susceptibility tests for Haemophilus

Answer 55

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

Question 56

Susceptibility tests for N. gonorrhoeae

Answer 56

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

Question 57

The E-Test

Answer 57

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

Question 58

E-test procedure

Answer 58

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)

Question 59

Kirby bauer results vs E-test results

Answer 59

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