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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
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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

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3
Q

this is critical when doing antimicrobial susceptibility testing (inoculum)

A

inoculum size

- particularly for testing susceptibility to penicillin and sulfonamides

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4
Q

constituents of culture media can either ______ or ______ antibiotic activity

A

enhance or antagonize

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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
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6
Q

alkaline pH

A

enhances activity of:

  • aminoglycosides
  • erythromycin
  • other macrolides
  • lincomycins
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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
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8
Q

susceptibility test media widely used in NA

A

Mueller-Hinton Agar

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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
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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
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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
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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
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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
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14
Q

diffusion rates depend on…

A 
solubility
molecular size
polarity fo the antibiotic
the growth medium
 the temperature chosen for incubation
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15
Q

diffusion rates depend on…

A 
solubility
molecular size
polarity fo the antibiotic
the growth medium
 the temperature chosen for incubation
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16
Q

growth rates depend on…

A

organism
nature of growth medium
temperature and atmosphere of incubation

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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
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18
Q

who establishes ZOI sizes

A

National Committee for Clinical laboratory standards (NCCLS)

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19
Q

Advantages of Kirby-Bauer

A
  • flexible (antibiotics)

- techinically simple

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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
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21
Q

Kirby-Bauer system requires supplementation with a … for situations where quantitative results are needed

A

dilution system

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22
Q

thick plate (how does this affect antimicrobial susceptibility testing)

A

smaller ZOI b/c won’t diffuse too far -> invalid!

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23
Q

thin plate (how does this affect antimicrobial susceptibility testing)

A

larger ZOI b/c antibiotic will diffuse faster!

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24
Q

heavy inoculum (how does this affect antimicrobial susceptibility testing)

A

not standardized; smaller ZOI

25
Q

light inoculum (how does this affect antimicrobial susceptibility testing)

A
  • bigger ZOI
  • uneven streaking
  • fuzzy margins
  • can’t read zones
26
Q

expired discs (how does this affect antimicrobial susceptibility testing)

A

smaller zone

- antibiotic not as potent

27
Q

mixed culture (how does this affect antimicrobial susceptibility testing)

A

invalid! NEED pure

28
Q

too many discs (how does this affect antimicrobial susceptibility testing)

A
  • too crowded; can’t measure
  • if overlapping zones but distinct edge = valid! measure radius
  • measure from smallest zone!!!
29
Q

wrong media (how does this affect antimicrobial susceptibility testing)

A

invalid test

doesn’t meet guideline criteria

30
Q

anaerobic incubation (how does this affect antimicrobial susceptibility testing)

A

invalid

KB can’t be used for anaerobes

31
Q

Staphylococci antibiotics

A 
P10
VA30
E15
SXT25
FOX
KZ30
DAZ
32
Q

P. aerginosa antibiotics

A 
CAZ30
CIP5
CN10
PRL100
TOB10
33
Q

Fastidious organisms will not grow on Mueller-Hinton unless it is …

A

supplemented with required growth factors

34
Q

T or F. Disc tests must be modified and standardized to fit each microorganism

35
Q

Kirby-Bauer may not be used for organisms grown on…

A

supplemented media, cultures incubated under increased CO2, organisms that grow more slowly than Enterics, pseudomonads, and staph

36
Q

H. influenzae isolates from critically ill patients are screened for this

A

beta-lactamase production

- using colonies from primary cultures

37
Q

medium for disc testing of Haemophilus

A

Haemophilus Test Medium (HTM)

  • M-HA supplemented wth bovine haematin, NAD, and yeast extract
  • 5-7% CO2 for 16-18 hrs
38
Q

common resistance mechanism of H. influenzae to ampicillin

A

beta-lactamase production

39
Q

PPNG testing method

A
  • 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
Q

a strain of S. pneumoniae with low, but significant resistance to penicillin is increasing

A
  • 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
Q

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 …

A

1 ug oxacillin disc

  • penicillin-susceptible = ZOI >20mm
  • penicillin-rsistant or relatively resistant ZOI <19 mm
42
Q

procedure for Pneumococci screen test

A
  • 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
Q

lab methods for detection of beta-lactamases include

A

iodometric
acidimetric
chromogenic cephalosporin
- positive results rapid with Haemophilus influenzae and GC; much longer with staph

44
Q

Many ________ B-lactamases are inducible

A

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
Q

contrasting to staph, H. influenzae and N. gonorrhoea are __________ th their B-lactamase production

A

constitutive

- synthesized continuously

46
Q

some gram negative bacteria like (________ ________) produce beta-lactamases that are cell-bound

A

B. fragilis

  • will not be detected in whole cell preparation
  • snification of whole cell preparation may be necessary for detection of B-lactanmases
47
Q

T or F. B-lactamase tests should only be performed on pure cultures

A

T!

always test with negative and psotiive control strains and staph should be pre-induced

48
Q

two chromogenic cephalosporins are available:

A

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
Q

the compounds most sensitive for detecting beta-lactamase producing strains of N. gonorrhoeae and H. influenzae

A

Chromogenic cephalosporin method: nitrocefin and pyridine-2-aco-p-dimethylaniline

50
Q

This is effective for detecting b-lactamases in staph

A

nitrocefin

51
Q

acidimetric methods for beta-lactamase testing

A
  • 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
Q

these are used to determine the minimum inhibitory concentration of antimicrobial agents

A

dilution methods

53
Q

MIC

A

the lowest concentration o antibiotic that will inhibit visible growth of a microorganism (ug/L)

54
Q

MIC

A

the lowest concentration o antibiotic that will inhibit visible growth of a microorganism (ug/L)

55
Q

dilution method of susceptibility testing

A
  • 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
Q

Macro-broth dilution susceptibility testing

A
  • 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
Q

Micro-broth dilution method of susceptibility testing

A
  • 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
Q

MBC

A
  • 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
Q

antibiotic gradient strip that gives a precise and accurate determination of MIC

A

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

MLSCI 242 (39 decks)

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