VTEC 2015 presentation given in Boston, on the use of long read sequencing technology to investigate an outbreak of STEC O157

1. The use of PacBio sequencing
to investigate phenotypic
changes during an outbreak of
STEC O157:H7
Lauren Cowley, PhD student @laurencowley4

2. STEC O157 phage typing scheme
• Used in the UK to type the
different strains and some
phage types(PT) are associated
with more severe disease than
others.
• Scheme based on the use of 16
bacteriophage that produce a
profile of phage infection for the
strain based on what level of
lysis is achieved by each phage
(Ahmed et al. 1987) and has
been used to categorize
outbreaks and sporadic cases.
2 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7

3. STEC O157
• STEC outbreak occurred in august 2012 in Belfast
• 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
• This change in PT confounded the epidemiological investigation and
characterisation of the outbreak strains.
3 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7

4. STEC O157
• There were no other clusters/outbreaks of this phage type related to food in
Northern Ireland, the UK or Europe at the time of the October 2012
outbreak.
• Chopped parsley garnish associated with illness
• Colindale collection typing indicated an association with North Africa or the
Eastern Mediterranean
4 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7

5. 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
• This indicated 2 separate introductions from a source population.
• BEAST analysis of STEC sequencing has shown us that we can expect ~3
SNPs per year so the last common ancestor of these strains is likely to be
~1 year previous to the outbreak
• The change from PT8 to PT54 is the gain of resistance to the previously
characterised group 3 typing phages (4, 5 and 14) so potentially only one
genetic event is involved
5 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7

6. PacBio Sequencing
• Illumina assemblies of STEC O157 normally produce high contig numbers
with prophage regions not being resolved
• Long read sequencing provides reads that span highly repetitive phage
regions so better single contig assemblies can be achieved
• Long read 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
6 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7

7. 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
7 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7

8. 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.
8 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7

9. 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
9 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7
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10. 10 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7

11. PT54
unique
H12
plasmid
11 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7

12. Phenotypic evidence of plasmid gain
Plasmid prep performed at Colindale that revealed 6 of the PT54 strains also
had 200kbp plasmid that was missing from all of the PT8 strains
12 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7
39R861545454 5454 548
8
8 88 88

13. Phenotypic evidence of plasmid gain
• All of the PT8 shared an AMR profile of:
AMP8/SUL256/STR8/TET8/TMP2
• All of the PT54 shared an AMR profile of:
AMP8/CHL8/CHL16/SUL256/STR8/TET8/TMP2
• Added plasmid-encoded chloramphenicol resistance for PT54 strain
• Indicative that all PT54 strains had this plasmid
• Suspected that plasmid is responsible for PT change
13 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7

14. Conjugation of plasmid switches the PT
Plasmid conjugation performed by David Gally’s Lab
14 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7
PT8 PT54PT54

15. Conclusions
• The use of PacBio sequencing has enabled a much more in depth view of
gene differences between 2 closely related strains of different PT
• Greater than expected rate of gene gain and loss that is likely to have
contributed to the phenotypic change in PT
• Genes of interest found on plasmid, including restriction methylase and inner
membrane genes, previous evidence of plasmid gain changing PT
• Work undertaken by DG’s lab to conjugate the plasmid into the PT8 strain
converted it to become PT54
• It had previously been assumed that closely phylogenetically related outbreak
strains would not vary so much in gene content but our analysis has indicated
that a relatively high rate of genetic variation can occur.
15 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7

16. Conclusions
• For complex bacterial genomes such as STEC O157:H7 differences in the
accessory genomes could only be fully understood using single molecule
sequencing technologies such as PacBio
• This outbreak provides insight into micro-evolutionary events that are
occurring in sink populations of pathogens.
16 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7

17. 17 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7
Acknowledgements
• Tim Dallman
• David L. Gally
• Jim Bono
• Claire Jenkins
• Martin Day
• Philip Ashton
• John Wain
• Kathie A. Grant
• Public Health Agency, NI
• Belfast City Council (Legal
Services)
• Belfast Health and Social Care
Trust
• Michael Wright
• Vivienne do Nascimento
• Neil Perry
• Yoshini Taylor
• Dawn Hedges
• Danielle Hall
• Martha Valencia
• Laura Stock
• Food Standards Agency
• Sean McAteer

18. Questions?
18 The use of PacBio sequencing to investigate phenotypic changes during an outbreak of STEC O157:H7