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Beyond Phylogeny:
Evolutionary analysis of
a mosaic pathogen
Dr Rosalind Harding
Departments of Zoology and Statistics,
Oxford University,UK
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
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
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
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, lactosenegative (5 atypical characteristics for the species).
Severe soft tissue
infections/
Necrotizing fasciitis
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.
Initial genetic analysis
 MLST: multi-locus sequence typing
 Sequences of fragments of ‘housekeeping’ genes
(dN/dS 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)
76
UPGMA tree of
concatenated
sequences of
10 genes: two
major groups:
I & II, plus ST8
97
78
94
I
99
80
11- Environment (Denmark)
65- Environment (Germany)
13- Environment (Denmark)
19- Human (USA)
59- Healthy fish (Israel)
17- Oyster (USA)
49- Environment (Germany)
44- Environment (Germany)
6- Diseased eels (Spain, Japan, Sweden, Taiwan),a
12- Environment (USA)
66- Environment (Germany)
9- Diseased eels (Denmark),b
47- Sea water (Japan)
41- Oyster (USA)
62- Oyster (USA)
35- Oyster (USA)
15- 1(Environment), 1(human) (USA)
51- Oyster (USA)
24- Oyster (USA)
29- Human (USA)
43- Human (Germany)
26- Oyster (USA)
28- Oyster (USA)
39- Oyster (USA)
48- Diseased eel (Denmark)b
53- Oyster (USA)
63- Oyster (USA)
10- Diseased eel (Denmark)b, healthy fish (Israel)
30- Oyster (USA)
38- Oyster (USA)
31- Oyster (USA)
52- Human (USA)
27- Oyster (USA)
34- Oyster (USA)
23- Oyster (USA)
54- Oyster (USA)
25- Oyster (USA)
4- Environment (USA)
3- Human (USA)
16- Human (USA)
22- Oyster (USA)
8- Human (61), healthy fish (1) (Israel)
84
88
II
70
72
77
84
100
82
98
99
0.002
45-Environment (Germany)
57- Human (Spain)
14- Environment (Denmark)
61- Human (Sweden)
69- Shrimp (Indonesia)
70- Human (Sweden)
1- Human (USA)
2- Human (USA)
56- Human (South Korea)
68- Human (Sweden)
55- Human (Singapore)
46- Human (Japan)
YJ016- Human (China)
18- Human (USA)
67- Human (Japan)
40- Human (USA)
32- Human (4), oyster (1) (USA)
42- Human (USA)
5- Environment (Spain)
58- Healthy fish (Israel)
20- Human (USA)
50- Human (Singapore)
7- Shrimp (Thailand)
CMCP6- Human (South Korea)
60- Oyster (USA)
21- Human (USA)
64- Human (USA)
36- Human (USA)
33- Human (USA)
37- Human (USA)
Vibrio
parahemolyticus
ST8=Biotype 3
All Biotype 3
isolates were
identical at level
of MLST
resolution.
Genetic differentiation into two ‘populations’ is not
explained by geographic location of isolates
Output from
STRUCTURE
analysis,
assuming K= 3
populations
Genetic differentiation into two ‘populations’ is
not explained by biotype distribution.
However, Biotype 3
does have a
distinctive
intermediate genetic
identity between the
populations.
Output from
STRUCTURE
analysis,
assuming K= 3
populations
Biotype 1 occurs
in both
populations
Biotype 3
Two populations: different disease associations
Population B is
associated with
disease in humans
Population A is
associated with
eel disease
Output from
STRUCTURE
analysis,
assuming K= 3
populations
UPGMA
Group II
UPGMA
Group I
Inferred ancestry
Biotype 3 is a hybrid between parents
from Population A and Population B
Biotype 3 is a mosaic genome
A
I
II
B
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.
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

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
Recombination exchange
between groups I & II is rare
Splits graph of allelic sequences from glp gene
I
ST8 (Biotype 3)
has a glp allele
from Population
B/group II
II
Alleles 12 and 38 from
Cluster II STs are more
closely related to Cluster I
Recombination rates within genes within
groups are high
 Evidence of recombination from Beagle:
www.stats.ox.ac.uk/~lyngsoe/beagle
Ancestral history is not as
simple as a tree.
Splits graph of alleles from
dtdS gene
II
I
Minimum of 9
recombination
events
Next Question.
Polymorphism for a complex trait?
 Is the genetic differentiation related to
pathogenicity phenotype?

higher odds for causing either human or eel
disease
Isolation in a metapopulation?
Is the genetic
differentiation
caused by
isolation between
populations?
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.
USA-Env
USA-ENV
Denmark-EEL
Israel-Env
Denmark-Env
Baltic Sea
USA-Env
USA-clinical
USA-Env
USA-Env
USA-Clinical
USA-Env
Baltic sea
Baltic sea
Japan-EEL
Denmark-eel
Denmark-Env
USA-Env
Japan-Env
USA-Env
Germany-Clinical
USA-Env
USA-Env
USA-Env
USA-Env
Denmark-EEL
USA-Env
USA-Env
USA-Clinical
USA-Env
USA-Env
USA-Env
USA-Env
USA-Env
Baltic sea
USA-Env
USA-Env
USA-eNV
USA-Clinical
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
USA-Clinical
USA-Clinical
USA-Clinical
Thailand-Env
USA-Clinical
USA-Clinical
USA-Clinical
Japan-Clinical
Taiwan-Clinical
USA-Clinical
Singapore-Clinical
Sweden-Clinical
S. Korea-Clinical
USA-Clinical
USA-Clinical
0.005
UPGMA group I
(Population A)
Biotype 3
In Biotype 3, genes
1, 2, 4, & 10 are
from group II, i.e.
human disease
associated.
UPGMA group II
(Population B)
The same split is preserved across
genes 1, 2, 4 & 10
1. Large chromosome: glp
4. Large chromosome: metG
2. Large chromosome: gyrB
10. Small chromosome: tnaA
But the same split is also preserved
across the other 6 genes, e.g.
5. Large chromosome: purM
8. Small chromosome: pntA
9. Small chromosome: pyrC
6. Small chromosome: dtdS
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
 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