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.’

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

Main 2nd year achievements

• Paper in review• TraDIS• Long read and short read outbreak analysis• Stx2a prophage affecting PT change

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.

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.

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

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

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.

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

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

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

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

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

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.

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

Prophage variation

PT54 unique plasmid

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

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

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 Stx2aTransposase Repressor Protein Cl regulatory proteinhypothetical protein Phage protein serine/threonine kinaseIS2 repressor TnpA Insertion element IS629 12kDa protein S4062hypothetical protein Transposase for insertion sequence element IS629hypothetical protein Transposase Integrase core domain protein

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