Antimicrobial sensitivity testing L12 Flashcards
what does clinical antibiotic-sensitivity testing tell us
Confirms the organism is sensitive to the antibiotic being used to treat the patient; or suggests an alternative antibiotic
what does epidemiological antibiotic-sensitivity testing tell us
Sensitivity data on common pathogens provides useful public health information, locally and nationally
what does pharmaceutical antibiotic-sensitivity testing tell us
Provides information on the activity of new antibiotics against a range of pathogens
what are the antibiotic-sensitivity methods
clinical
epidemiological
pharmaceutical
what are the phenotypic methods
agar diffusion assays
broth dilution assays
agar incorporation assays
what happens in an agar diffusion assays
antibiotic diffuses from an impregnated paper disc into an agar medium on which the test organism is growing
what happens in a broth dilution assay
Serial dilutions of the antibiotic in broth growth medium are inoculated with the test organism
what happens in an agar incorporated assay
antibiotic dilutions are incorporated in an agar medium and spot inoculated with a number of test organisms
how is an agar diffusion assay carried out
- Spread plate (blood agar) with lawn of test strain
- Place antibiotic discs on lawn
- Incubate overnight
- Examine plate for zones of inhibition
how can agar diffusion assays be sped up
using an antibiotic disc applicator, is automated applies all the antibiotics at once
what is the radius of the zone of inhibition determined by
- rate of antibiotic diffusion
- growth rate of the bacteria – incubation temperature
- sensitivity of the bacteria to the antibiotic
what is MIC
Minimum Inhibitory Concentration
what does the zone of inhibition relate to
size of the zone of inhibition is related to the MIC of the test organism
what controls are important in agar diffusion assays
- control organism of known sensitivity
- inoculate test organism and control organism on the same “Stokes Assay”
- standardise the methods used (EUCAST)
for staphylococcus aureus what zone size indicates it is resistant
<20mm (MRSA)
for staphylococcus aureus what zone size indicates it is sensitive
> 20mm (MSSA)
what are the problems with agar diffusion assays
- low solubility, ionic charge, high molecular weight
- growth rate of test bacteria
- antibiotic concentration in the disc
what is the problem with low solubility, ionic charge, high molecular weight in agar diffusion assay
poor diffusion = small zone of inhibition, even if the organism is sensitive
what is the problem with slow growing bacteria
slow growing organisms give rise to large zones, can diffuse out a long way before organism grows to be visible
slower growth period it can be inhibited more effectively as lower numbers of colonies on plate
how can the problem of antibiotic concentration in the disc in agar diffusion assays be solved
Commercially available discs have a tolerance of 67-150%; a 30micrograms disc could have 20-45micrograms
how can the MIC be determined
E test
broth dilution assay
agar incorporation
what is the E test
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
how is the MIC determined in the E test
E Test meniscus - the MIC corresponds to the point where the bacterial growth crosses the numbered strip.
what happens in a broth dilution assay
Doubling dilutions of antibiotic in 1ml of broth medium
Inoculate each tube with 105 cells of the test strain and incubate overnight
how is the MIC found in broth dilution assay
determine the lowest concentration which inhibits growth
what is MBC
minimum bactericidal conentration
how can the MBC be found in broth dilution assay
lowestconcentrationof antibiotic that kills, not just inhibits bacteria
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
what is bactericidal
kills bacteria
what is bacteriostatic
stops bacteria growing, doesn’t kill it
which is higher MBC or MIC
MBC usually higher
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
how can MIC be found from agar incorporation assay
use series of selected antibiotic (break-point) concentrations, put differentconcentrationsof antibiotics on plate and test sensitivity
One plate with no antibiotic to see if bacteria grows
Changingconcentrationof antibiotics in agar can see levels of resistance
MIC’s can also be estimated
what factors affect sensivity tests
slow growing organisms
inoculum size must be standardised
composition of culture medium
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
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
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
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
what are the advantages of agar diffusion
low technology
quick to set up
cheap
flexible
what are the disadvantages of agar diffusion
expensive in time and material when large numbers of cultures are used
what are the advantages of broth dilution
accurate MIC is obtained
automation is easy
what are the disadvantages of broth dilution
Expensive in time and materials when large numbers of cultures are used
what are the advantages of agar incoporation
Twenty or more strains on one plate
easy to automate
what are the disadvantages of agar incorporation
Antibiotic stability in agar
antibiotic concentration is critical
what are examples of automated systems
Vitek
TREK Sensititre
Dade Behring Walkaway system
what us synergy
get better effect than just one antibiotic, one antibiotic will help the other one work better
what is antagonism
one antibiotic stops the other from working, allowing bacteria to grow between that area
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
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
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
what is an example of synergy - protect the other from in activation
inhibitor of beta-lactamase enzymes (clavulanic acid) protects a beta-lactam (amoxycillin)
what is an example of synergy - over come spontaneous mutation to resistance
combination therapy of three drugs for TB treatment
what may occur in the antagonism of antibiotics
- antagonism of bactericidal activity
- inducible resistance
- chemical interactions
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
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
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
what do antibiotic sensitivity tests detect
phenotype; the ability to grow in the presence of an antibiotic
what is an alternative to antibiotic sensitivity testing
detect the protein product of the resistance gene; i.e. PBP 2’ gene product in MRSA
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
where is the mecA gene found
in an SCCmec element inserted in the orfX gene in S.aureus genome
how many SCCmec elements are there
at least 8 types
how are the SCCmec elements detected
can detect all with several PCR primer pairs
what is MRSA
meticillin-resistant Staphylococcus aureus
what causes penicillin resistance in MRSA
mecA gene, makes a protein that can work effectively even with antibiotic present
what is an alternative sensitivity test to phenotypic detection
detect the resistance gene itself; i.e. mecA gene in methicillin resistant S.aureus (MRSA)
what test can be used to detect the protein product of mecA
latex agglutination test
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
