module 2

Question 1

subtyping methods

Answer 1

serotype, phage typing, RFLP, AFLP, PFGE, MLST, WGS

Question 2

what is serotyping

Answer 2

testing if bacteria have the same surface antigens.
H = flagella
K = capsule
O = LPS

Question 3

method for serotyping

Answer 3

latex beads coated with antibody. bacteria is stirred in. if there is an antigen - antibody interaction then agglutination will occur.
this can confirm or rule out if a isolate belongs to a particular serogroup

Question 4

what is phage typing

Answer 4

testing the relation of strains through their susceptibility to bacteriophages. bacteriophages have different mechanisms, and therefore different molecular targets. this means strains with genetic differences will have a differential phage susceptibility

Question 5

method for phage typing

Answer 5

entire plate of bacterial lawn divided up. inoculate each square with a unique phage. zone of lysis indicates susceptibility to the phage.

Question 6

what is a restriction fragment length polymorphism

Answer 6

tests for genetic variations between bacterial isolates

Question 7

method for RFLP

Answer 7

isolate and purify DNA from bacterial isolate. treat with restriction enzymes which correspond to short, repetitive sequences. separate fragments by gel electrophoresis. transfer gel to a filter membrane. probe against insertion sequences, or another repetitive region.
difference in cleavage sites = different bands

Question 8

disadvantage for RFLP and AFLP

Answer 8

do not test specific genetic variations, i.e. you do not know where the mutation is and what it is

Question 9

what is AFLP for

Answer 9

testing for genetic variations between bacterial isolates

Question 10

method for AFLP

Answer 10

digest DNA with restriction enzymes which cut frequently. ligate with end specific adapters. amplification of fragments using PCR. analyse using gel electrophoresis and examine bands. difference in bands = prob not related

Question 11

what is PFGE for

Answer 11

tests how closely isolates are. commonly used for foodborne bacteria

Question 12

method of PFGE

Answer 12

use restriction enzymes which do not cut often. gives large fragments of DNA. these must be separated on a gel with an alternating current.
a polymorphism at the restriction cut side will be shown through a loss of bands. indicates further relation

Question 13

what is MLST for

Answer 13

looks for mutations in highly conserved genes – usually these mutations are synonymous

Question 14

MLST method

Answer 14

amplify housekeeping/highly conserved genes. sequence by sanger sequencing. identify alleles. assign a sequence type

Question 15

disadvantages of MLST

Answer 15

use of highly conserved/housekeeping genes often fails to detect variability of closely related strains.
sequencing of 7 genes is relatively expensive and time consuming

Question 16

WGS pros and cons

Answer 16

PROS:
- know exactly which genes are involved
- know the mutations
- higher resolution than other subtyping methods
CONS:
- relatively slow and expensive