Antimicrobial susceptibility assays & molecular assays

why test for antimicrobial susceptiblity?

• Know which antibiotics can or especially can't be used for treatment / experiments

• Assess which dose may be required for treatment

• Epidemiology (spread of resistance)

tests for antimicrobial susceptiblity: phentoypic tests

• Disc diffusion assay

• Broth dilution test (MIC test)

• E-test

broth dilution test

• incubation in presence of a concentration range of the antibiotic

• MIC (minimal inhibitory concentration): lowest concentration of antibiotic that inhibits growth

tells you exactly at what point the bacteria becomes resistant

you’re looking for the point the growth stops

quantifiable test, a lot of work

E-test

• strip with predefined antibiotic gradient

prepare bacteria culture on petri dish

in the middle you lay this drip (top pic)

the strip has gradient of concentratiosn of antibiotic - higher numbers = higher doses

starts diffusing int othe medium

highest conc would spread furthest and kill more - until it cant kill anymore cos it gets diluted as it spreads out

MIC (Minimum Inhibitory Concentration) would be between 1.5 and 2 where inhibition stops

can put multiple ones on same plate - just make sure diff antibiotics are far enough away from each other to not react with each other

disc diffusion assay

same idea as E test but no gradient

take plates, prepare same way, but get paper disks with antibiotics - if you get large inhibition zone = dead bacteria/cant grow/susceptible to the antibiotic = not resistant

if they dont get killed then zonewould be small/non existent so they are resistant

practical setup of disc diffusion assay

• plate

• bacterial suspension (E. coli)

• pipette

• spreader

• forceps

• discs impregnated with antibiotic

3 discs per plate provided, with:

• Penicillin G (P10)

• Ertapenem (ETP10)

• Ciprofloxacin (CIP5) or Tetracycline (TE10)

Tests for antimicrobial susceptibility: pros and cons

Advantages phenotypic tests:

• proves resistance/susceptibility

• Test for multiple antimicrobials in single experiment

• Some tests give MIC value > informs treatment

Disadvantages phenotypic tests:

• slow > maybe too slow in specific cases

• requires culture available

Molecular/genomics-based methods for antimicrobial resistance detection

Tests for antimicrobial susceptibility: genotypic tests

PCR

Disadvantages for rapid diagnostics:

• sensitive for contamination

• requires special (expensive) thermocycler

• can be difficult to multiplex, not robust

LAMP (Loop-mediated isothermal Amplification)

isothermal - temp doesnt change

• simple DNA extraction method (one lysis and absorbance step)

• PCR at one temperature (65 or 67 degrees) using 4 primer pairs

• visual detection by green fluorescence or measurement of tubidity by real-time turbidimeter

(Loop-mediated isothermal Amplification) LAMP

pic compares lamp to pcr

advantages of LAMP

• more rapid and (semi)quantitative

• less-specialised equipment requirement

• can be used with easy detection systems

— On-site/on-farm rapid detection

— Strip-based colorimetric detection

genotypic tests AMR (antimicrobial resistance)

Advantages genotypic tests:

• highly specific

• rapid

• culture-independent

Disadvantages genotypic tests:

• does not prove resistance

• does not inform about MIC

• needs separate test for each target

Use of genomics / genome sequences in AMR & drug discovery

AMR

• Large collections of genomes nowadays available

— (Genbank, PubMLST, Enterobase, other repositories)

• Can be used for predicting antimicrobial resistance genes

• Genotypic predictions may need subsequent validation

example data obtained

• Pornsukarom et al, BMC Genomics 2018

• Salmonella enterica isolates

typed for:

• serotype

• 6 types of antibiotic

resistances

• Check for correlations

between AMR types

potential weaknesses of all computational methods

Based on search algorithms and scores

• May miss point mutations

• Minor sequence differences may have major functional implications!

Presence of gene(s) does not mean functionality

• Expression levels, other genes/metabolites required?

Still 90-99% correlation found in different studies/bacteria!

ALL THESE ANALYSES REQUIRE APPROPRIATE VALIDATION!