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34 Cards in this Set

  • Front
  • Back
microbial characterisation
pure culture techniques: for selecting for particular organisms (diff. plates)
agar based plates

DNA fingerprinting generated by digestion/ electrophoresis
allows distinguish/ compare between different samples

antigenic typing: ID strains at a fine level by typing

Next-gen DNA seq
single species or complex mix

Metabolic Typing: different reactions
16S rRNA gene
was used by Woese and Fox to discover Archaea

can investigate taxonimc relations

variable and hypervariable regions evolve in a faster rate (mosaic structure)

total length 1500bp long
variable region

red colours high variation
blue is low variation

varaible regions have different lengths
laerger teh size the more variaton (information)

microbial characterisation
-> specific species
-> complex communities

primers to constant regions (should bind to most organisms) amplify variable ones -> seq/ fingerprinting to identify

design primers that bind to variable (specific species)
-> either get a band or not, depending on whether they are present or not.

case study:
each band corresponds to a diferrent species
badn corresponds to
food poisoning
salmonella 10,000 per anum

campylobacter 70,000 per anum

e. coli 1,000
norovirus (enteric disease)
gram negative pathogens
campylobacter jejuni
salomnella enterica
e. coli
vibrio cholerae
yersinia enterolitica
shigella spp (e.coli like)
gram positive pathogens
staphylococcus aureus
clostridium perfringes
clostridium botulinum
bacillus cereus
listeria monocytogenes
non bacterial pathogens
aspergillus

aflatoxins

viruses
bacterial strategies
non invasive: 0157 e'coli attach to intestin. cells rearragnge cell surface structure=> allows them to persist

intracellular pathogens=> hide from competitin and avoid phagocytosis
detection
homogenise food (buffere and broth)=> enrich homogenate for organisms being tested for (pathogens) , =? differ. plating + presumptive identification (colour change on plate) => more specific typing (serotyping etc)

bypass all culture steps => sequence directly form sample
techiques and level of accuracy
typing => species level
important to ID on subspecies level (pathogenic strains)

reference strains
Detection
determination of presence or absence of a particular food poisoning bacterium in sample
Identification
determination of a detected species as belonging to a known group (genus, species, strain) by any technique
Typing
the use of techniques to distinguish isolates at the strain/ subspecies level

reference strain can be very useful
Typing techinques
serotyping
phage typing
antibiograms (resistance)
mol. characterisation
- plasmid dna
- genotyping

- physical/ exterior/ specific (Fla typing, Penner and Lior typing)
- physiology (secretion systems)
- metabolism of the cell (capacity) species level
- DNA based methods => strains
core genome, dispensable genome (strain level)
small SNPs and 16S rRNA (PFGE, AFLP), whole genome sequencing
Traditional and modern methods
selective media, gram stains, colorimetric
=> genus level

visual methods ( fluorescent microscopy

biochemical rests

Immunological rests (immunomagnetic separation, serotyping)

DNA based
PCR, RFLP, AFLP (amplified)

nex gen
- need for one that does not require known bit of genome (for identifying unknown)
commercial pathogen detection systems
ELISA,
API 20E
standard ID system

campylobacter

dehydrated substrate ion tube
grow organism
colour change, depending on hat can/cannot be metabolised
good for gram-ive
not so useful for capmylobacter due to limited metabolic capacity
20 markers!
+/- points added up for groups of three
score is read and compared to table
limitations: uncharacterised metabolisms
MAST scheme
for Camylobacter jejuni

narrow down to species level
3 markers
Campylobacter serotyping

Penner scheme
O-serotypes

capsular polysacharhyde being recoginsed

uses HEAT STABLE antigen (present after heat treatment)

Guilian Barre syndrome is related to Campylobacter (some seroptypes)

Heat up suspension
extract proteins (expose to EDTA)
42 serotypes of C. jejuni and 18 for C.coli


different sugars
inner and outer portion of lipopolysaccharides

step 1) extract heat stable polysaccharides by exposing to saline solution at 100 oC or EDTA
2) treated cells are exposed to erythrocytes (have lipoglycoprotein receptor which binds to the antigen)

3) antigen-sensitised erythrocytes dispensed into wells containing antiserum, in two-fold dilutions

*specific antibodies for each serum type. antibodies bind to polysaccharides (which are also bound to the erythrocytes) causing the haemaglutination, signalling the detection of the specific serotype

4) incubated overnight at 37C
=> highest dilution showing haemaglutination is recorder as the titre
underlying genetic variation
genes responsible for production of lipopolysaccharydes vary in number and type
-> result in different structure
genetic variation can be detected by serotyping scheme
Campylobacter serotyping

Lior scheme
heat-Labile antigens. 21 serotypes recognised
now, more than 100 serotypes!

rapid slide agglutination technique
1) loop of live bacteria mixed with drop of antiserum on glass slide
2) asses agglutination after 30-45 sec (bacteria clump together if the antigen is recognised by the antiserum)
Molecular typing
PCR, RFLP, AFLP, PFGE, MLST, microarray, protein array
PCR
y
RFLP
variation in patterns after cutting by a specific RE
AFLP
amplify a specific gene followed by RFLP
PFGE
pulse field gel electrophoresis
large fragments at seperation
cut up entire chromosome with enzyme that cuts rarely=> separate large fragemtns on special gell
MLST
multilocous sequence typing

sequence specific regions and look for variation in them
microarray/ protein array, next gen seq
y
PCR
standard
nested : second seet of primers amplify first fragments more sensitive, detects rarer pathogens
multiplex PCR: multiple primers in same tube, diff. bands according to organisms present

quantitative: product threshold level.
RFLP on Salmonella
enteriditis and typhimurium

give different patterns in closely related species
must be a variable region (like 16S)
RFLP with fla gene
flaA or flaA and flaB

picks up different strains of Campylobacter

detect proteins with antibodies or on DNA level
MLST
house keeping genes

evolve slower than virulence genes
allocate SNPs at different groups

evolutionary relationships and clonality extent
evolution of chromosome (scattered)

changes are usually silent (changes on the last position of the codon)

different disease related to different

marker choice criteria:
-chromosomal location
- suitability for primer design
- sequence diversity
Campylobacter jejuni MLST
Dingle et al 2001

7 loci, 194 isolates
=> 155 sequence types ==> 62 clonal lineages

selection against amino acid change
next gen seq
more sequences

no need to clone, start from mix and separate out

cost per base is much less (human genome for 1000$)

in microbiology


+++ high trhoughput (many food samples at the same time)
+safer bench work
+insights on taxonomy and activities
rareaction
steeper-> more species

community less diverse as meat spoilt-> gives insight in process