Summary of work done in first 2 years of PhD

1. End of 2nd
year review
Lauren Cowley
‘The use of genome sequencing to investigate the molecular basis of bacteria-phage
interaction of the Escherichia coli O157 typing phages and the elucidation of the
biological and public health significance of phage type.’

2. Ebola deployment and PhD
extension
• I have just returned from a month in Sierra
Leone working in the PHE ebola diagnostic
labs
• I have successfully extended my PhD stipend
by a month to cover this and so will now finish
on January 5th
2016

3. Main 2nd
year achievements
• Paper in review
• TraDIS
• Long read and short read outbreak analysis
• Stx2a prophage affecting PT change

4. Typing phage sequencing
• Paper detailing results of the sequencing of
the STEC O157 typing phages has been
reviewed at BMC genomics and minor
revisions were requested. These sections will
be revised and the paper will be re-submitted
by February 5th
.
• Results enabled us to group the phages into 4
groups that correlated their sequence
similarity with their reactivity profiles.

5. TraDIS
• Library production on strain 1465 (2a/c –ve
9000) to select for phage susceptibility and
resistance genes is making good progress;
• 1 Transposon per 50bp have inserted and this
means there are ~20 per gene
• The insertions are evenly spaced and you
don’t see insertions in essential genes such as
gyrA and rpoB.

7. TraDIS
• However we would like to increase the
number of insertions to 1 every 25bp as the
phage resistance and susceptibility genes are
so unknown.
• I will increase this number over the next 3
months and will subsequently move on to
testing the library with phage selections

8. Phage selections
• Selection 1 will take the PT32 stx knockout
library and subject it to typing phage 13 that
should produce clear lysis and look for an
increase in insertions in genes that enable
phage resistance
• Selection 2 will take the same library and
subject it to typing phage 12 that it is resistant
to and look for a decrease in insertions in
genes that enable phage infection

9. Second paper preparation
• I am in the process of preparing a paper on
the comparison of long read and short read
sequencing results when looking into an
outbreak.
• I aim to finish this paper and submit it to a
journal within the next six months.

10. Two Closely related strains in an
outbreak… How close is close…

11. Belfast outbreak
• STEC outbreak occurred in Belfast in august
2012
• Six cases of PT8 were linked to a specific food
outlet
• Six weeks later, 150 cases of PT54 were
associated with the same food outlet
• The change from PT8 to PT54 is the gain of
resistance to the group 3 phages (4, 5 and 14)
so potentially only one genetic event is
involved

12. Illumina sequencing
• Phylogenetic analysis performed on the core
genome of the strains using a mapping
technique against Sakai
• The PT8 and PT54 strains were found to be
only 3 SNPs apart

13. PacBio Sequencing
• PacBio sequencing of one PT8 and one PT54
strain revealed a far greater degree of
variation
• The two strains were shown to have 31 SNPs
between them in fully aligned regions using
the alignment program NUCMER
• This still does not include the accessory
regions of each strain

14. Accessory variation
• Analysis of prophage, plasmid and total gene
content differences was undertaken
• Genes present in one strain and not the other
would not align so would not be included in
the total SNP count
• A script was written in python to blast
annotated genes from each PacBio sequenced
strain against all genes in the other strain and
if no hits were found the gene was recognised
as unique to that strain

15. Gene variation
• The PT8 strain was found to have 153 unique
genes.
• The PT54 strain was found to have 96 unique
genes.
• The PT54 strain also had an additional 220
genes encoded on a large (240kbp) plasmid
that was not found in the PT8 strain.

16. Prophage variation
• The program PHAST was used to look at the
prophage regions in the two strains
• The prophage regions had not previously been
analysed from the illumina sequencing as they
had not assembled
• There were 11 shared prophage regions
between the strains
• 6 unique to the PT54 strain and 7 unique to
the PT8 strain

17. Prophage variation

18. PT54 unique plasmid

19. Plasmid work
• Previous evidence of plasmid gain changing PT
• Genes of interest found on plasmid, including
restriction methylase and inner membrane
genes
• Work to be undertaken in David’s lab to
conjugate the plasmid into the PT8 strain and
see if this changes it to become PT54

20. Loss of Stx2a affecting PT
Roslin Ref Description GBRU Ref Phage type
9000 Original PT21/28 IPRAVE isolate, stx2a & stx2c H144660654 21/28
10671 Original PT32 IPRAVE isolate, stx2c only H144660655 32
1456 9000 with stx2c phage partly deleted H144660656 21/28
1460 9000 with stx2a phage entirely deleted H144660657 32
1463 10671 with stx2c phage entirely deleted H144660658 32
1465 1456 with stx2a phage entirely deleted H144660659 32
1467 1460 with stx2c phage partly deleted H144660660 32
1599 9000 with stx2a & stx2c genes entirely deleted H144660661 21/28

21. Comparing Stx2a prophages
• Comparison of Stx2a prophage in strain 16438
(PT32 2a/2c) to strain 9000 (PT21/28 2a/2c),
very closely related strains phylogenetically
• Backbone of prophage largely similar only vary
in a few genes;
Only on 16438 Stx2a Different in 16438/9000 Stx2a Only on 9000 Stx2a
Transposase Repressor Protein Cl regulatory protein
hypothetical protein Phage protein serine/threonine kinase
IS2 repressor TnpA Insertion element IS629 12kDa protein S4062
hypothetical protein Transposase for insertion sequence element IS629
hypothetical protein
Transposase
Integrase core domain protein

22. Future work
• Complete production of denser TraDIS library
and perform selections
• Undertake plasmid conjugation in David’s lab
to try to switch the PT and submit outbreak
paper
• Further characterise Stx2a prophage genes to
find genes involved in PT switch