1. Beyond Phylogeny: Evolutionary analysis of a mosaic pathogen Dr Rosalind Harding Departments of Zoology and Statistics, Oxford University,UK

2. Research Collaborators Naiel Bisharat Dept of Epidemiology and Preventative Medicine, Tel Aviv University, Israel Derrick Crook Nuffield Dept of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, UK Martin Maiden Dept of Zoology, University of Oxford Bisharat et al. (2005) Hybrid Vibrio vulnificus Emerg Infect Dis 11:30-35

3. Population Genetics Interplay of micro-evolutionary processes Mutation and recombination Population structure and demography Natural selection Questions and strategy concern: Understanding steady-state patterns of diversity Learning about ancestral history (genealogy) Understanding dynamics: emergence of new strains Major technical problem Trees don’t show recombination events

4. Vibrio vulnificus Globally wide-spread inhabitant of marine and estuarine environments Dangerous waterborne pathogen: case fatality rate for V. vulnificus septicemia may reach 50% Typically, cases of V. vulnificus infection are sporadic Human infection acquired through eating contaminated raw or undercooked sea food, or via contamination of wounds by seawater or marine animals

5. Disease Outbreak in Israel Major outbreak of systemic V. vulnificus infection among fish market workers and fish consumers Epidemiology 1995: first case 1996: 32 patients 1997: 30 patients all handled fresh Tilapia fish cultivated in inland fish farms 1998: marketing policy changed to prevent sale & handling of live Tilapia fish New biotype identified Distinctive biochemistry, eg salicin-negative, lactose- negative (5 atypical characteristics for the species).

6. Severe soft tissue infections/ Necrotizing fasciitis

7. V. vulnificus diversity Biotype 1: sampled from environment, healthy fish, shellfish etc; associated with sporadic human infection Biotype 2: associated with disease in eels Biotype 3: new cause of human disease outbreak in Israel. Where did Biotype 3 come from? Biotypes have been defined based on biochemical tests of phenotype.

8. Initial genetic analysis MLST: multi-locus sequence typing Sequences of fragments of ‘housekeeping’ genes (d N /d S ratios < 1.0) 10 genes, 5 from each of the two chromosomes, each fragment ~400 bp Concatenated sequence of 4,326 bp defines sequence types (STs) Isolates: Biotype 1: n=82 isolates (39 from human disease, 43 from environment Biotype 2: n=15 isolates (13 from eels) Biotype 3: n=61 isolates (60 from human disease, 1 from fish-pond water)

9. UPGMA tree of concatenated sequences of 10 genes: two major groups: I & II, plus ST8 I ST8=Biotype 3 All Biotype 3 isolates were identical at level of MLST resolution.

10. Output from STRUCTURE analysis, assuming K= 3 populations Genetic differentiation into two ‘populations’ is not explained by geographic location of isolates

11. Genetic differentiation into two ‘populations’ is not explained by biotype distribution. Output from STRUCTURE analysis, assuming K= 3 populations Biotype 3 Biotype 1 occurs in both populations However, Biotype 3 does have a distinctive intermediate genetic identity between the populations.

12. Output from STRUCTURE analysis, assuming K= 3 populations UPGMA Group I UPGMA Group II Two populations: different disease associations Population B is associated with disease in humans Population A is associated with eel disease

13. Biotype 3 is a hybrid between parents from Population A and Population B Inferred ancestry

14. I II A B Biotype 3 is a mosaic genome

15. Clonal expansion of Biotype 3 Maynard Smith, J et al (2000) BioEssays 22:1115-1122 Disease outbreak clones emerge from a background of low frequency variation connected by mutation and recombination.

16. Progress summary The disease outbreak in Israel (Biotype 3) was caused by a clonal expansion of Sequence Type 8 ST 8 is a mosaic sequence created by recombination between parents from Populations A and B Next questions How much recombination? How did the genetic differentiation between Populations A and B arise? Population A = UPGMA Group I = Eel disease associated Population B = UPGMA Group II = Human disease associated

17. Splits graph of concatenated sequences from 10 genes Cluster I = Population A Association with eel disease (biotype 2) ST8 = Biotype 3 Cluster II = Population B Association with human disease

18. Recombination exchange between groups I & II is rare ST8 (Biotype 3) has a glp allele from Population B/group II Alleles 12 and 38 from Cluster II STs are more closely related to Cluster I Splits graph of allelic sequences from glp gene II I

19. Recombination rates within genes within groups are high Minimum of 9 recombination events Ancestral history is not as simple as a tree. II I Evidence of recombination from Beagle: www.stats.ox.ac.uk/~lyngsoe/beagle Splits graph of alleles from dtdS gene

20. Polymorphism for a complex trait? Is the genetic differentiation related to pathogenicity phenotype? higher odds for causing either human or eel disease Next Question.

21. Isolation in a metapopulation? Is the genetic differentiation caused by isolation between populations?

22. Any clues from diversity in individual genes? If polymorphism, perhaps expect differentiation to localise to one or a subset of genes? If differentiation is due to isolation between populations, expect all genes to show the same patterns.

23. USA-Env USA-ENV Denmark-EEL Israel-Env Denmark-Env Baltic Sea USA-Env USA-clinic al USA-Env USA-Clinical USA-Env Baltic sea Japan-EEL Denmark-eel Denmark-Env USA-Env Japan-Env USA-Env Germany-Clinical USA-Env Denmark-EEL USA-Env USA-Clinical USA-Env Baltic sea USA-Env USA-eNV USA-Clinical USA-Env Israel-Clinical Denmark-Env Spain-Clinical Baltic sea Sweden-Clinical USA-Env - S.Korea-Clinical Japan-Clinical Indonesia-Env USA-Clinical Israel-Env Spain-eel farm Singapore-Clinical USA-Clinical Sweden-Clinical USA-Clinical Thailand-Env USA-Clinical Japan-Clinical Taiwan-Clinica l USA-Clinical Singapore-Clinical Sweden-Clinical S. Korea-Clinical USA-Clinical 0.005 Biotype 3 UPGMA group I (Population A) UPGMA group II (Population B) In Biotype 3, genes 1, 2, 4, & 10 are from group II, i.e. human disease associated.

24. The same split is preserved across genes 1, 2, 4 & 10 1. Large chromosome: glp 2. Large chromosome: gyrB 4. Large chromosome: metG 10. Small chromosome: tnaA

25. But the same split is also preserved across the other 6 genes, e.g. 6. Small chromosome: dtdS 9. Small chromosome: pyrC 5. Large chromosome: purM 8. Small chromosome: pntA

26. Conclusions Differentiation between populations is evident across all 10 genes. Recombination exchange between populations is rare across all genes. Within populations: Large numbers of alleles related through recombination as well as mutation history Isolation by distance? Polymorphism? Recombination is key to generating diversity in Vibrio vulnificus

28. Clonal Expansion In expansions of clonal complexes, new mutations are evident before recombination. (Linkage disequilibrium due to selective sweep.) Differentiation is shaped by selection: clonal complexes emerge as new adaptations Meta-population structure Old population diversity generated by mutation and recombination is sustained. Differentiation is shaped by isolation: outbreaks emerge as new recombinants