Lecture 16 - Bacterial typing and molecular epidemiology Flashcards
(24 cards)
define bacterial typing
identifying and categorising strains based on their relationships
what are the 4 typing classes of bacteria?
- clonal
- highly related = all 3 genomes mostly same
- mod related = core same
- unrelated = all 3 genomes different
define bacterial species
related strains with conserved core genome + varied dispensable/unique genes
what are the pros and cons of phenotyping?
- pros = rapid, cheap, quick
- cons = can’t discriminate strains, genes affected by environ = inaccurate measure of genetic distance
How are genera/species identified by phenotyping?
dichotomous tree of positive/negative results
explain how an API test works and its pros and cons
- Analytical profile index (API)
- wells with dehydrated substrates to detect enzymes/fermentation of carbs/aa’s/proteins
- pros = quick, cheap, easy
- cons = can’t discrimate strains
define epidemiology
study of distribution, frequency and determinants of disease in population over time/locations
what is molecular typing using for in epidemiology?
outbreak investigation = related cases, causative strain, patient and environmental isolate matchings
in epidemiology what does the same strain vs related strains found in isolates mean?
- same = same source
- related = going thru infected people = getting less virulent
define propagaed epidemic
gradual increase (above expected) then gradual decline from host-to-host transmission from 1 infected person
what are the 2 uses of molecular typing
- genetic lineage prediction = evolutionary relationships
- characterisation = identify types needing investigation
explain pulsed field gel electrophoresis (PFGE)
- electrophoresis to resolve fragments 50-2000kB
- requires restriction fragment length polymorphism (RFLP) = different band patterns
- no. of changes = evolutionary distance
describe the method of PFGE
- prep = pure, wash, embed in agarose
- lyse with detergent and proteinase = DNA into gel
- RE digest with low frequency cutter SmaI
- PFGE with 3 electrodes and 2-3 long runs
- EtBr + UV
how is PFGE different from electrophoresis? discuss the pros and cons
- electro = high frequency RE (4bp) = tiny bands with poor resolution
- PFGE = low frequency RE (6-8bp) = big bands with good resolution
- pros = resolution, whole genome visualised, gold standard
- cons = expensive, training, long runs
explain ribotyping
- RFLP + southern blot = digest, separate, membrane with probes
- detect 16S and 23S rRNA genes/intergenic regions
- conserved ribosomal DNA thruout genome = same probe different bacteria
Describe the method of ribotyping
- prep = pure, wash
- lyse and purify DNA = 90-100C, detergent, proteinase, phenol/chloroform
- RE with high frequency cutter EcoRI
- electrophoresis
- southern blot
- autoradiography/chemiluminscence visualisation
what are the pros and cons of ribotyping?
- pros = reproducible, possible automation
- cons = less discriminatory than PFGE bc fewer bands, no whole genome analysis
explain randomly amplified polymorphic DNA (RAPD)
- PCR = 10bp random primers + low annealing temp = random hybridising separated by electro by band intensity
- banding patterns = insertions/deletions between primer binding
describe the method of RAPD
- prep = pure, wash
- lyse = 90-100C, detergent, proteinase
- purify = phenol/chloroform
- PCR = 10bp random primers + 35C annealing
- electrophoresis
- EtBr + UV
what are the pros and cons of RAPD? what is it best used for?
- pros = cheap, efficient, sensitive
- cons = reproducibility, standardisation, band intensity varies
- rapid typing and distinction of unrelated isolates
explain multilocus sequence typing (MLST)
- 7-8 dispersed house-keeping genes selected and PCR for different alleles
- sequence type (ST) for each isolate with all allele numbers
- 400-500bp fragments sequenced
what are the pros and cons of MLST?
- pros = unambiguous, reproducible, scalable, automatable, easily transferred/compared
- cons = expensive, limited databases
explain whole genome sequencing (WGS)
- Entire genome of isolate for $80 in a few hrs
- compare all alleles @ every locus in phylogenetic tree
what are the pros and cons of WGS?
- pros =unambiguous, reproducible, scalable, automatable, easily transferred/compared
- cons = limited databases, skilled labour, AI replaced, MGEs skew phylogenetic trees = need bioinformatics
