Bacterial Diversity

Question 1

What are the non molecular notable differences between E.coli and Shigella?

Answer 1

E.coli is motile and commensal, Shigella is non motile and pathogenic

Question 2

What type of comparison was used for E.coli and Shigella in the pre-molecular era?

Answer 2

Sero-typing

Question 3

What can Sero-typing tell you about a microorganism?

Answer 3

Serotyping utilises immune recognition of different surface antigens to distinguish different strains of bacteria.
Bacteria of the same serotype will cross react to the same antibodies

Question 4

Other than serotyping, what pre-molecular study was used to compare bacteria?

Answer 4

DNA hybridisation

How well do DNA extracts from different genomes hybridise?

Question 5

During Early molecular studies of bacterial diversity, did their initial findings correlate well with serotyping?

Answer 5

No no non no they did not. silly billies

eg MLEE data and serotype data

Question 6

How was electrophoresis used in early molecular studies of bacterial diversity?

Answer 6

Electropheresis was used to measure the motility of different enzymes from different E.coli strains

Question 7

What could multi-locus enzyme electrophoresis (MLEE) tell you about bacteria?

Answer 7

produced quantitative molecular data which can be used to look at the evolutionary relationships between strains

Question 8

What was the first sequencing technology used in bacterial diversity studies?

Answer 8

PCR amplification of single genes , genes sequenced and compared

Question 9

What was seen when E.coli and Shigella (individual) gene sequences were compared

Answer 9

There was convergent evolution and diversity between strains.
Convergent evolution could be used to explain the biochemical diversity seen in serotyping

Question 10

What information did phylogenetic trees provide for bacterial diversity studies?

Answer 10

Evolutionary signals for different genes in the same genomes
Suggested RECOMBINATON (if I don't say this I lose) of genes over time - where organisms had acquired genes from different strains

Question 11

What is MLST and what is it used for?

Answer 11

Multi-locus sequence typing

Used to compare multiple different gene loci at once

Question 12

What type of genes were used in MLST studies?

Answer 12

Housekeeping genes of roughly 400bp

Question 13

What did MLST show in terms of pathogenetic bacterial diversity?

Answer 13

Showed that pathogenic strains of the same pathotype had often inherited virulence and toxicity genes from different donors

Question 14

What are examples of pathogenicity donors?

Answer 14

Plasmids

prophages

Question 15

Why is 16srRNA useful for bacterial diversity studies?

Answer 15

16srRNA is found in all bacteria
Is a highly important molecule so evolves very slowly.
Can be used to compare bacteria of completely different species (not just gena)

Question 16

What part of the 16srRNA is sequenced to use for comparison studies?

Answer 16

variable regions ‘V Loops’

Rest of the sequence is highly conserved

Question 17

How is 16srRNA profiling carried out?

Answer 17

Universal primers to conserved regions are used to amplify the variable regions
Sequenced using Illumina, Pacbio or sanger

Question 18

What are the limitations with 16srRNA profiling?

Answer 18

Primers might not be truly universal - some sequences might not be 100% conserved
Contamination can be an issues (any contamination will bind universal primers and signals will be read)
Only useful for culturable bacterial species

Question 19

What can cause sequence error?

Answer 19

PCR bias, some sequences amplify better than others which can cause incorrect quantification of the number of species.
Most organisms have multiple copies of 16srRNA gene which can vary in sequence, causing an overestimation of the number of taxa present in a sample

Question 20

What is microbial dark matter?

Answer 20

99% of unculturable bacteria present in bacterial population of the WORLLLD

Question 21

What is the study called for bacterial diversity studies beyond 16s?

Answer 21

metagenomics

Question 22

What is metagenomics?

Answer 22

DNA extracted from an environmental sample, which is fragmented and sequenced

Question 23

What is Kraken used for?

Answer 23

To try and identify individual reads from deep sequencing of complex populations

Question 24

What was the problem with deep sequence analysis that occurred initially?

Answer 24

Big fat problemo - control sequence still found lots of sample reads (control was no sample put through but reagents)
meant reagents contained contaminants picked up as sequence reads
Poo Poo all this data was now fucked and not applicable

Question 25

What was found when next generation sequencing was used to sequence human microbiome?

Answer 25

Found that in different environments (eg mouth, stomach) there was an altered microbiome for disease phenotypes which were not obviously ‘catching’
(basically - altered microbiological profiles in associated diseased organs in patients than in normies - even though disease isn’t typically ‘bacterial’ - so cant be passed on from dutty hands etc)

Question 26

What is basic process of single cell genomics?

Answer 26

Use optical tweezers or other method to extract single cell of bacteria (part of microbial dark matter population)
and sequence using PCR

Question 27

What are the limitations of single cell genomics?

Answer 27

Can not get full genome as only using PCR so bias, and no repeats
coverage of genome will b patchy

Question 28

What is iCHIP?

Answer 28

Microfluidics technology used to try and bypass the issue of not microorganisms not being suited to lab conditions

Question 29

What is the make up of an iCHIP chamber?

Answer 29

Has agar in well and semipermeable membrane to environment

Question 30

How does iCHIP work?

Answer 30

Dilute a bacterial sample so there is roughly one cell per chamber
Put the iCHIP back in a fairly native environment so that isolated sample can culture enough genetic material to be used for sequencing