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Antimicrobial sensitivity testing L12 Flashcards

1
Q

what does clinical antibiotic-sensitivity testing tell us

A

Confirms the organism is sensitive to the antibiotic being used to treat the patient; or suggests an alternative antibiotic

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2
Q

what does epidemiological antibiotic-sensitivity testing tell us

A

Sensitivity data on common pathogens provides useful public health information, locally and nationally

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3
Q

what does pharmaceutical antibiotic-sensitivity testing tell us

A

Provides information on the activity of new antibiotics against a range of pathogens

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4
Q

what are the antibiotic-sensitivity methods

A

clinical
epidemiological
pharmaceutical

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5
Q

what are the phenotypic methods

A

agar diffusion assays
broth dilution assays
agar incorporation assays

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6
Q

what happens in an agar diffusion assays

A

antibiotic diffuses from an impregnated paper disc into an agar medium on which the test organism is growing

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7
Q

what happens in a broth dilution assay

A

Serial dilutions of the antibiotic in broth growth medium are inoculated with the test organism

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8
Q

what happens in an agar incorporated assay

A

antibiotic dilutions are incorporated in an agar medium and spot inoculated with a number of test organisms

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9
Q

how is an agar diffusion assay carried out

A
  1. Spread plate (blood agar) with lawn of test strain
  2. Place antibiotic discs on lawn
  3. Incubate overnight
  4. Examine plate for zones of inhibition
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10
Q

how can agar diffusion assays be sped up

A

using an antibiotic disc applicator, is automated applies all the antibiotics at once

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11
Q

what is the radius of the zone of inhibition determined by

A
  • rate of antibiotic diffusion
  • growth rate of the bacteria – incubation temperature
  • sensitivity of the bacteria to the antibiotic
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12
Q

what is MIC

A

Minimum Inhibitory Concentration

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13
Q

what does the zone of inhibition relate to

A

size of the zone of inhibition is related to the MIC of the test organism

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14
Q

what controls are important in agar diffusion assays

A
  • control organism of known sensitivity
  • inoculate test organism and control organism on the same “Stokes Assay”
  • standardise the methods used (EUCAST)
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15
Q

for staphylococcus aureus what zone size indicates it is resistant

A

<20mm (MRSA)

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16
Q

for staphylococcus aureus what zone size indicates it is sensitive

A

> 20mm (MSSA)

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17
Q

what are the problems with agar diffusion assays

A
  • low solubility, ionic charge, high molecular weight
  • growth rate of test bacteria
  • antibiotic concentration in the disc
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18
Q

what is the problem with low solubility, ionic charge, high molecular weight in agar diffusion assay

A

poor diffusion = small zone of inhibition, even if the organism is sensitive

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19
Q

what is the problem with slow growing bacteria

A

slow growing organisms give rise to large zones, can diffuse out a long way before organism grows to be visible
slow​er growth period it can be inhibited more effectively as lower numbers of colonies on plate

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20
Q

how can the problem of antibiotic concentration in the disc in agar diffusion assays be solved

A

Commercially available discs have a tolerance of 67-150%; a 30micrograms disc could have 20-45micrograms

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21
Q

how can the MIC be determined

A

E test
broth dilution assay
agar incorporation

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22
Q

what is the E test

A

Plastic strips contain a gradient of antibiotic and are printed with the concentration
During incubation of the plate antibiotic diffuses out and sets up a gradient
higher concentration at top of strip

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23
Q

how is the MIC determined in the E test

A

E Test meniscus - the MIC corresponds to the point where the bacterial growth crosses the numbered strip.

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24
Q

what happens in a broth dilution assay

A

Doubling dilutions of antibiotic in 1ml of broth medium

Inoculate each tube with 105 cells of the test strain and incubate overnight

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25
how is the MIC found in broth dilution assay
determine the lowest concentration which inhibits growth
26
what is MBC
minimum bactericidal conentration
27
how can the MBC be found in broth dilution assay
lowest concentration of antibiotic that kills, not just inhibits bacteria ​
28
what control is done in broth dilution assay
Control organism of known sensitivity included in parallel | Can also be automated in microtitre dishes with a plate reader
29
what is bactericidal
kills bacteria
30
what is bacteriostatic
stops bacteria growing, doesn't kill it
31
which is higher MBC or MIC
MBC usually higher
32
what is the agar incorporation assay
Inoculate a plate containing antibiotic with a number of test cultures and controls, using a multipoint inoculator incubate overnight at 37°C Record cultures which show significant growth at that single antibiotic concentration (Breakpoint Method ) Automation is possible
33
how can MIC be found from agar incorporation assay
use series of selected antibiotic (break-point) concentrations, put different concentrations of antibiotics on plate and test sensitivity One plate with no antibiotic​ to see if bacteria grows Changing concentration of antibiotics in agar can see levels of resistance  MIC’s can also be estimated
34
what factors affect sensivity tests
slow growing organisms inoculum size must be standardised composition of culture medium
35
how is the assay of M. tuberculosis sped up
M. tuberculosis is a slow growing organism grow organism on media that contains radioactive carbon source, look for production of 14CO2 from a labelled substrate, if inhibited by antibiotic will not get radioactive carbon dioxide speed up assays by 1-2 weeks
36
what is the effect if put more bacteria on plate in inoculum
generally get smaller zones for the same organisms and antibiotics - antibiotic doesn't have time to diffuse properly
37
how is inoculum size standardised
always use the same density of cells - using a spectrophotometer or a device to ensure the density of the cultures are the same so know there is the same amount of bacteria on the plates
38
how does the composition of culture medium affect sensitivity testing
- thymidine interferes with sulphonamide detection - too much carbohydrate will lead to acid pH that affects aminoglycosides activity - divalent cations affect aminoglycosides
39
what are the advantages of agar diffusion
low technology quick to set up cheap flexible
40
what are the disadvantages of agar diffusion
expensive in time and material when large numbers of cultures are used
41
what are the advantages of broth dilution
accurate MIC is obtained | automation is easy
42
what are the disadvantages of broth dilution
Expensive in time and materials when large numbers of cultures are used
43
what are the advantages of agar incoporation
Twenty or more strains on one plate | easy to automate
44
what are the disadvantages of agar incorporation
Antibiotic stability in agar | antibiotic concentration is critical
45
what are examples of automated systems
Vitek TREK Sensititre Dade Behring Walkaway system
46
what us synergy
get better effect than just one antibiotic, one antibiotic will help the other one work better​
47
what is antagonism
one antibiotic stops the other from working, allowing bacteria to grow between that area ​
48
what may occur in the synergy of antibiotics
- potentiate the activity of the other at a biochemical level - assist the other to penetrate the bacterial cell - protect the other from inactivation - overcome spontaneous mutation to resistance
49
what is an example of synergy - potentiate the activity of the other at a biochemical level
Combination of trimethoprim and sulphonamides targets sequential steps in folate synthesis in bacteria
50
what is an example of synergy - assist the other to penetrate the bacterial cell
combinations of penicillin and aminoglycoside are effective against enterococci due to increased uptake of latter
51
what is an example of synergy - protect the other from in activation
inhibitor of beta-lactamase enzymes (clavulanic acid) protects a beta-lactam (amoxycillin)
52
what is an example of synergy - over come spontaneous mutation to resistance
combination therapy of three drugs for TB treatment
53
what may occur in the antagonism of antibiotics
- antagonism of bactericidal activity - inducible resistance - chemical interactions
54
what is an example of antagonism of bactericidal activity
bacteriostatic agents (tetracycline and chloramphenicol) can inhibit activity of bactericidal agents like beta-lactams that rely on bacterial growth for killing activity
55
what is an example of antagonism - inducible resistance
induction of chromosomal beta-lactamases by potent inducers like cefoxitin, may cause resistance to poor inducers like cefotaxime
56
what is an example of antagonism - chemical interactions
high concentrations, beta-lactam ring of some beta-lactams can interact chemically with amino-groups of aminoglycosides, inactivating both compounds
57
what do antibiotic sensitivity tests detect
phenotype; the ability to grow in the presence of an antibiotic
58
what is an alternative to antibiotic sensitivity testing
detect the protein product of the resistance gene; i.e. PBP 2’ gene product in MRSA
59
what is detected when detecting the gene product
Detects the protein product of the mecA resistance gene in MRSA If MRSA is present the latex will agglutinate into easily visible clumps
60
where is the mecA gene found
in an SCCmec element inserted in the orfX gene in S.aureus genome
61
how many SCCmec elements are there
at least 8 types
62
how are the SCCmec elements detected
can detect all with several PCR primer pairs
63
what is MRSA
meticillin-resistant Staphylococcus aureus
64
what causes penicillin resistance in MRSA
mecA gene, makes a protein that can work effectively even with antibiotic present
65
what is an alternative sensitivity test to phenotypic detection
detect the resistance gene itself; i.e. mecA gene in methicillin resistant S.aureus (MRSA)
66
what test can be used to detect the protein product of mecA
latex agglutination test
67
why are molecular methods better than phenotypic assays
in theory much faster as phenotypic assay needs 24hr growth over night before can look at the plates PCR assay kit designed to do it in an hour if have resistance gene or not - can deal with

Microorganisms + Disease (16 decks)

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