LAB - Antimicrobial Susceptibility Testing Flashcards
1
Q
Methods for determining susceptibility fall into three general classes:
A
- diffusion methods: Kirby-Bauer + mods
- dilution techniques: broth + agar
- detection of resistance mechanisms: B-lactamases
2
Q
In all of the antimicrobial susceptibility test methods, this is required for testing
A
pure culture
- several colonies are testing in order to ensure that test results are representative of the bacterial population
3
Q
this is critical when doing antimicrobial susceptibility testing (inoculum)
A
inoculum size
- particularly for testing susceptibility to penicillin and sulfonamides
4
Q
constituents of culture media can either ______ or ______ antibiotic activity
A
enhance or antagonize
5
Q
examples of culture media antagonists
A
- thymine/thymidine = end-products of folate metabolism; antagonistic to activity of trimethoprim and sulfonamides
- para-aminobenzoic acid (PABA) = structurally similar to the sulfonamides and will act as an antagonist to their activity
- divalent cations = antagonize cell entry of the aminoglycosides, cell binding of polymyxins or my chelate with tetracyclines
- increased phosphate concentrations = may reduce activity of aminoglycosides
6
Q
alkaline pH
A
enhances activity of:
- aminoglycosides
- erythromycin
- other macrolides
- lincomycins
7
Q
agar concentration and depth for antibiotic susceptibility testing
A
- affect diffusibility of antibiotics into agar
- must be controlled in diffusion tests
- recommended depth of agar for disc diffusions tests = 4 mm
8
Q
susceptibility test media widely used in NA
A
Mueller-Hinton Agar
9
Q
Mueller-Hinton agar
A
- low concentrations of PABA, thymine, thymidine
- may be used for testing sulfonamides and trimethoprim along with other antimicrobials
- broth formula = Ca++ and Mg++ to approximate physiological levels
> IF NOT, organisms tested may appear more susceptible to the aminoglycosides - agar = higher levels of cations and phosphates
- does NOT support the growth of fastidious bacteria
10
Q
temperature for antimicrobial susceptibility testing
A
- temperature = changes will alter rates of bacterial growth and antibiotic diffusion
- accepted std = 35C
- methicillin resistance staph may not be detectable at higher temps
11
Q
atmosphere for antimicrobial susceptibility testing
A
- incubate in room air
- 5-10% CO2 may reduce pH of culture media and thereby alter antibiotic activity
- anaerobic = prevent uptake of aminoglycosides
12
Q
antimicrobial agents incubation conditions for antimicrobial susceptibility testing
A
- storage at -20C
- current use = refrigerated
- desiccant used to keep discs and powders dry
13
Q
disc concentration for antimicrobial susceptibility testing
A
- total antibiotic per disc
- content is standardized and controlled by the FDA
- performance monitored daily; testing control strains of known susceptibility
14
Q
diffusion rates depend on…
A
solubility molecular size polarity fo the antibiotic the growth medium the temperature chosen for incubation
15
Q
diffusion rates depend on…
A
solubility molecular size polarity fo the antibiotic the growth medium the temperature chosen for incubation
16
Q
growth rates depend on…
A
organism
nature of growth medium
temperature and atmosphere of incubation
17
Q
Kirby-Bauer Method
A
- standardized for rapid growth bacteria
- MH agar
- standard inoculum, 10^8 CFU/mL
- inoculum density achieved by growing bacteria in TSB for several hours; adjusting turbidity to 0.5 McFarland standard!!
- adjusted broth - cotton swab - surface of MHA
- std antibiotic conctns in paper discs are applied to surface
- plate incubated within 15 mins (to avoid prediffusion)
- 35C
18
Q
who establishes ZOI sizes
A
National Committee for Clinical laboratory standards (NCCLS)
19
Q
Advantages of Kirby-Bauer
A
- flexible (antibiotics)
- techinically simple
20
Q
deficiencies of the Kirby-Bauer Method
A
- inapplicability to slower-growing bacteria, anaerobes
- non-quantitative interpretation
- inaccuracy in detecting slightly elevated levels of resistance
21
Q
Kirby-Bauer system requires supplementation with a … for situations where quantitative results are needed
A
dilution system
22
Q
thick plate (how does this affect antimicrobial susceptibility testing)
A
smaller ZOI b/c won’t diffuse too far -> invalid!
23
Q
thin plate (how does this affect antimicrobial susceptibility testing)
A
larger ZOI b/c antibiotic will diffuse faster!
24
Q
heavy inoculum (how does this affect antimicrobial susceptibility testing)
A
not standardized; smaller ZOI
25
light inoculum (how does this affect antimicrobial susceptibility testing)
- bigger ZOI
- uneven streaking
- fuzzy margins
- can't read zones
26
expired discs (how does this affect antimicrobial susceptibility testing)
smaller zone
| - antibiotic not as potent
27
mixed culture (how does this affect antimicrobial susceptibility testing)
invalid! NEED pure
28
too many discs (how does this affect antimicrobial susceptibility testing)
- too crowded; can't measure
- if overlapping zones but distinct edge = valid! measure radius
- measure from smallest zone!!!
29
wrong media (how does this affect antimicrobial susceptibility testing)
invalid test
| doesn't meet guideline criteria
30
anaerobic incubation (how does this affect antimicrobial susceptibility testing)
invalid
| KB can't be used for anaerobes
31
Staphylococci antibiotics
```
P10
VA30
E15
SXT25
FOX
KZ30
DAZ
```
32
P. aerginosa antibiotics
```
CAZ30
CIP5
CN10
PRL100
TOB10
```
33
Fastidious organisms will not grow on Mueller-Hinton unless it is ...
supplemented with required growth factors
34
T or F. Disc tests must be modified and standardized to fit each microorganism
T
35
Kirby-Bauer may not be used for organisms grown on...
supplemented media, cultures incubated under increased CO2, organisms that grow more slowly than Enterics, pseudomonads, and staph
36
H. influenzae isolates from critically ill patients are screened for this
beta-lactamase production
| - using colonies from primary cultures
37
medium for disc testing of Haemophilus
Haemophilus Test Medium (HTM)
- M-HA supplemented wth bovine haematin, NAD, and yeast extract
- 5-7% CO2 for 16-18 hrs
38
common resistance mechanism of H. influenzae to ampicillin
beta-lactamase production
39
PPNG testing method
- Penicillinase-producing N. gonorrhoea tested for B-lactamse (colonies directly from plate_
- disc test on GC agar base + 1% Isovitaltex
- 20-24 hrs at 35C in 5-7% CO2
- <19 mm zones if positive
- Spectinomycin
- agar dilution method for other strains (NOT B-lactamase)
40
a strain of S. pneumoniae with low, but significant resistance to penicillin is increasing
- mechanismo f type of resistance is chromosomallly medited alteriation of PBPs
- nothing to do with B-lactamase
MIC <0.06 ug per L = susceptible
MIC 0.12-1 ug per L = relatively resistant
MIC >2 uug per L = resistant
41
Pneumococcal isolates from CSf, blood, and other body fluids may be screened for low level resistance to penicillin by a disc diffusion test using a ...
1 ug oxacillin disc
- penicillin-susceptible = ZOI >20mm
- penicillin-rsistant or relatively resistant ZOI <19 mm
42
procedure for Pneumococci screen test
- TSB = 0.5 McFarland standard
- M-H agar supplemented witj 5% sheep blood
- 1 ug oxacillin disc
- incubate at 35C for 18-24 hrs in 5-10% CO2
* * some strains of pneumococci require CO2 for initial isolation; Oxacillin results do not appear to be affected by CO2 incubation **
- if Pen Resistant Relatively.. then do a regular KB with certain antibiotics
43
lab methods for detection of beta-lactamases include
iodometric
acidimetric
chromogenic cephalosporin
- positive results rapid with Haemophilus influenzae and GC; much longer with staph
44
Many ________ B-lactamases are inducible
staphylococcal
- small quantities
- detectable quantities only after repeated exposure to antibiotic
- staph may give pos results sooner if PRE-induced with a beta-lactam
45
contrasting to staph, H. influenzae and N. gonorrhoea are __________ th their B-lactamase production
constitutive
| - synthesized continuously
46
some gram negative bacteria like (________ ________) produce beta-lactamases that are cell-bound
B. fragilis
- will not be detected in whole cell preparation
- snification of whole cell preparation may be necessary for detection of B-lactanmases
47
T or F. B-lactamase tests should only be performed on pure cultures
T!
| always test with negative and psotiive control strains and staph should be pre-induced
48
two chromogenic cephalosporins are available:
nitrocefin (yellow to red)
PADAC or pyridine-2-azo-p-dimethylaniline cephalosporin (purple, colurless, then yellow)
**BOTH exhibit a rapid colour change when the amide bond of the beta-lactam is hydrolyzed by a beta-lactamase**
49
the compounds most sensitive for detecting beta-lactamase producing strains of N. gonorrhoeae and H. influenzae
Chromogenic cephalosporin method: nitrocefin and pyridine-2-aco-p-dimethylaniline
50
This is effective for detecting b-lactamases in staph
nitrocefin
51
acidimetric methods for beta-lactamase testing
- uses penicillin as a substrate
- causes penicillin to be broken down to penicilloic acid
- different indicators used to detect acid production
- ex: phenol red to change form red to yellow
52
these are used to determine the minimum inhibitory concentration of antimicrobial agents
dilution methods
53
MIC
the lowest concentration o antibiotic that will inhibit visible growth of a microorganism (ug/L)
54
MIC
the lowest concentration o antibiotic that will inhibit visible growth of a microorganism (ug/L)
55
dilution method of susceptibility testing
- serial two-fold dilution concentrations to broth or agar
- approximations of those concentrations that are attainable in vivo
- twofold inherent error
- some MICs may lie close to one of the test concentrations resulting in a 1 dilution variation from the 'true' MIC
56
Macro-broth dilution susceptibility testing
- antimicrobial agent serially diluted in Mueller-Hinton broth medium containing standard inoculum of an organism at a final concentration of 10^8 CFU/L
- MIC = lowest concentration of antibiotic that inhibits macroscopically visible growth (last clear tube)
- a control always added (known MIC)
- MIC of a test can only be reported if control results are acceptable
- precise; reference methods (reserved for these)
57
Micro-broth dilution method of susceptibility testing
- small volume versions of concentional tube dilution techniques
- 0.1 ml rather than 1.0 mL
- 10^4 CFU in the 0.1 mL of antibiotic solution
- determine MICs
- same inherent error as macro
58
MBC
- minimum bactericidal concentratin
- done in combination with MIC dilution methods
- can be determined by subculturing aliquots of tubes showing no visible growth and examining for the endpoint that shows 99.9% kill
- smallest concentration of antibiotic which on subculture fails to grow or has 99.9% decrease in growth
59
antibiotic gradient strip that gives a precise and accurate determination of MIC
E-test
- can be used with most microorganisms including anaerobes and other fastidious bacteria
- MICscale is continuous; broad concentration range covering 15-fold dilutions
- combination of concepts of dilution and diffusion tests
- more precise
- similar process as disc diffusion test BUT
- different bc uses preformed and stable antibiotic concentration gradient
