1.9. Microbial typing techniques

Question 1

Microbial typing:

Answer 1

identification of a microorganism to a strain level

Question 2

strain

Answer 2

a population of bacteria presumed to descend from a single bacterium

Question 3

phenotype

Answer 3

physical characteristic
e.g. antigens and proteins expressed, biochemical reactions, growth requirements, resistance to antibiotics, resistance to bacteriophages

Question 4

genotype

Answer 4

the nucleotide sequence found on the chromosome of the organism

Question 5

why use microbial typing?

Answer 5

helps identification of epidemiologically linked isolates: same or different

Question 6

Serotyping

Answer 6

• Based on antigenic determinants of bacterial cell components
• Outer membrane, flagella, capsule
• Useful in certain organisms only e.g. Salmonella, Shigella, Streptococcus pneumoniae

Question 7

Molecular typing techniques are divided into

Answer 7

1. genome restriction analysis
2. PCR based typing
3. sequence based typing

Question 8

Genome restriction analysis

Answer 8

• DNA is cut into pieces using specific enzymes and the lengths/fragments analysed on a gel/membrane
• Pulse Field Gel Electrophoresis (PFGE)

Question 9

PCR based typing

Answer 9

• Specific DNA regions are amplified by PCR and analysed on a gel
• Amplified fragment length polymorphism (AFLP)

Question 10

Sequence based typing

Answer 10

• Determination of DNA sequence
• Multi Locus Sequence Typing (MLST)
• Whole genome Sequencing (WGS)

Question 11

Pulse-field gel electrophoresis (PFGE)

Answer 11

1. gold standard method
2. organsims embedded in agarose gel, cells are lysed and chromosomal DNA is released
   - digested with restriction enzymes
   - DNA fragments separated by electrophoresis (using pulsed current) –> pattern based on sizes of the DNA fragments
3. commonly used method
4. highly reproducible and discriminatory
5. may be difficult to interpret
   - i.e. significance of differences in banding profile

Question 12

Pros of PFGE

Answer 12

Gold standard for many bacteria due to:

1. highest degree of discrimination between strains (overall good discrimination power)
2. standardized protocols for many different bacteria

Question 13

cons of PFGE

Answer 13

labour intensive:

1. expensive (equipment and restriction enzymes for high discriminatory power)
2. ambiguous band scoring (variable signla intensities, background noise, differential band mobility, gel distortions)
3. takes several days
4. insufficient discriminative power for some isolates, others not typable

Question 14

WGS

Answer 14

• Whole genome sequencing
• process of determining the complete DNA sequence of an organism’s genome at a single time
  •This is process is performed using Next Generation Sequencing Technology
  •Allows us to differentiate between organisms with a precision that other typing methods do not allow
  •Facilitates better tracking of outbreaks or emerging lineages within a community/or country

Question 15

what is the advantage of having WGS data?

Answer 15

• can use bioinformatics –> can perform many analyses using various different computer programs/ softwares:
  1. serotyping
  2. antimicrobial resistance typing
  3. MLST
  4. virulence genes