1. Application of Whole Genome Sequence for both
Characterising and Studying Phylogenetic Structure of
Salmonella enterica serovar Weltevreden
Slide 1
Tirzania Frannetta Sopacua (zgv589)
Supervisor:
Marianne Halberg Larsen
Anders Dalsgaard
3. Introduction
Slide 3
• Salmonella animal origin
• Rarely found in aquaculture increase
occurrence
• Aquaculture Asian countries food
safety awareness
• Aquaculture contamination fecal-oral
4. Slide 4
Human = Seafood
Human ≠ Seafood
S. Weltevreden
Additional
GI-related to
carbohydrate?
Additional
Gene?
Chitinolytic
Activity
5. Slide 5
PFGE
lack power to distinguish closely related overestimate clonal
point mutation restriction site different band
WGS :
Higher discriminatory genetic relatedness
High Similar Band in
PFGE
Characterizing
Epidemiology or phylogenetic structure
6. Objectives
Slide 6
1. Applying WGS typing method in twelve strains of S. Weltevreden
previously isolated from shrimp farms in Vietnam and tilapia farm in
China
2. Generating genotype data such as AMR, plasmid, and SPI from WGS data
3. Studying phylogenetic structure within twelve S. Weltevreden strains and
global context with including other strains which are available in NCBI
from different BioProject (PRJEB1397, PRJNA161807, and PRJNA20591).
4. Comparing metabolic pathways present in S. Weltevreden genomes with
pathways present/absent in other Salmonella serovars in order to find
specific pathways for S. Weltevrede
5. Characterizing S. Weltevreden strains for some relevant phenotypic traits
based on the genome sequences
8. Results and Discussion
Slide 8
Strain
Name
Country Source Year
Genome
Size
Contigs n50
85C China Tilapia 2013 4.798.643 599 17.107
75C China Tilapia 2013 4.746.952 527 19.538
62C China Tilapia 2013 4.562.188 567 16.825
30C China Tilapia 2013 4.709.829 605 17.102
28C China Tilapia 2013 4.732.751 515 18.284
24C China Tilapia 2013 4.805.833 478 22.325
95V Vietnam Shrimp 2013 4.920.739 473 27.373
74V Vietnam Shrimp 2013 4.881.470 480 23.399
30V Vietnam Shrimp 2013 4.855.353 545 20.597
28V Vietnam Shrimp 2013 5.001.617 505 24.097
13V Vietnam Shrimp 2013 4.860.326 927 12.155
3V Vietnam Shrimp 2013 5.047.682 429 30.928
1. The Genome Statistics
• High number of contigs and lower number of n50
workable but not a high quality data
• Vietnam strains have slightly bigger genome size than
China strains
9. Slide 9
2. The Results of WGS Analysis
Strain
Name
Country Source Year
Resistance
(phenotyping)
Resistance
Gene
Class 1
Integron
Plasmid SPI
3V Vietnam
Shrimp
(Intensive)
2013 S Not Found Not Found IncFII(S) 3, 13, 14, C63PI
13V Vietnam
Shrimp
(Extensive)
2013 FFC, TMP, CHL Not Found Not Found IncFII(S) 13, 14, C63PI
95V Vietnam
Shrimp
(Extensive)
2013 SMX Not Found Not Found Not Found 3, 13, 14, C63PI
28V Vietnam
Shrimp
(Extensive)
2013 S Not Found Not Found IncFII (S) 9, 13, 14, C63PI
30V Vietnam
Shrimp
(Extensive)
2013 CHL Not Found Not Found
IncFII(S),
Col(MGD2)
13, 14, C63PI
74V Vietnam
Shrimp
(Extensive)
2013 S Not Found Not Found IncFII (S)
3, 13, 14, C63PI
62C China
Tilapia
(Integrated)
2013 SMX, TMP Not Found Not Found Not Found 13, 14, C63PI
28C China
Tilapia (Non-
Integrated)
2013 SMX,TMP Not Found Not Found Not Found 3, 13, 14, C63PI
75C China
Tilapia (Non-
Integrated)
2013 SMX,TMP,TET
strA,strB,sul2
,tet(A)
In IncQ1
Plasmid
IncFII(S),
IncQ1
13, 14, C63PI
30C China
Tilapia (Non-
Integrated)
2013 SMX,TMP Not Found Not Found Col(MGD2) 13, 14, C63PI
24C China
Tilapia (Non-
Integrated)
2013 SMX, TMP Not Found Not Found Not Found 9, 13, 14, C63PI
85C China
Tilapia (Non-
Integrated)
2013 SMX,TMP Not Found Not Found IncFII(S) 13, 14, C63PI
Repeat AMR
experiment
New Plasmid?
14. 4. Metabolic Pathways
• Based on BioCyc database
Carbohydrate
Metabolism
Biosynthesis
Degradation
NO SIGNIFICANCE DIFFERENCE
between S. Weltevreden and other
serovar related to possible aquatic
survival
15. • The Predicated Function of GI Related To
Carbohydrate and Orthologs
• Genes in GIs were assessed individually interesting
finding from this analysis is the product of SENTW_319
i.e. fructose-1,6-bisphosphatase which helps
cyanobacteria to withstand unfavorable condition in the
aquatic environment
• Orthologs common and unique protein
• 2826 proteins that shared between the strains
• 300 proteins are predicted unique for S. Weltevreden
whereas:
-193 are hypothetical protein
- 107 proteins involving in a different function.
16. • Transporters
Transporters
S. Agona
str.
SL483
S. Enteritidis str.
P125109
S. Typhimurium str.
LT2
S. Weltevreden str.
2007-60-3289-1
Uptake transporters 77 77 100 150
Transporters assigned to
transport reactions
77 77 100 150
Genes assigned to transport
proteins
77 77 100 150
All transported substrates 89 71 76 101
Substance Transport System
polar amino acid:
L-arginine
L-aspartate
L-glutamate
L-glutamine
L-histidine
L-lysine
L-ornithine
arginine transport ATP-binding protein ArtP
ATP-binding component of histidine transport
ATP-binding protein of glutamate/aspartate transport system
glutamine transport ATP-binding protein GlnQ
putative ATP-binding component of a transport system
a polyamine
ABC superfamily (atp_bind), spermidine/putrescine transporter, ATP-binding component of
putrescine transport system, putrescine/spermidine ABC transporter ATPase,
Spermidine/putrescine import ATP-binding protein potA
quaternary amine:
betaine aldehyde
betaine aldehydehydrate
choline
choline sulfate
γ-butyrobetaine
glycine betaine
glycine betaine/L-proline transport ATP-binding protein
an Fe(III)-siderophore
ferric dicitrate
ABC transporter ATP-binding protein
ATP-binding component of hydroxymate-dependent iron transport
ferric enterobactin transport ATP-binding protein FepC
Fe3+ ABC transporter, ATP-binding protein
putative ABC transport ATP-binding protein
• Specific for S. Weltevreden:
17. Slide 17
• Based on RAST
Our strains showed high similarity
analysis from Biocyc can be used
1) PTS mannose specific,
mannitol-specific and
cellobiose-specific
2) CRISPR protein associated with
unknown function,
3) aerobic C4-dicarboxylate transporter
for fumarate, L-malate, D-malate,
succinate,
4) CMP-N-acetylneuraminate-beta-
galactosamide-alpha-2,3-
sialyltransferase,
5) cytolethal distending toxin subunit B,
6) helicase, C-terminal:DEAD/DEAH box
helicase, N-terminal,
7) tetracycline efflux protein (TetA),
8) cyclohexadienyl dehydratase,
9) octaprenyl diphosphate synthase,
9) type I restriction-modification system,
DNA-methyltransferase subunit M,
10) fructose-1,6-
bisphosphatase, type I,
11) 4'-phosphopantetheinyl transferase
(EC 2.7.8.-) [enterobactin] siderophore,
and
12) chitinase.
18. 5. In Vitro Experiment
• Chitinolytic activity
S. Weltevreden lack of
chitinolytic activity
19. • Growth Experiment with Different Salt Concentration
• Increasing the salt content will induce different growth response
• No different between S. Weltevreden and S. Typhimurium LT2
20. • Growth Experiment with Different Sole Carbon Source
0.001
0.01
0.1
1
0 3 6 9 12 15 18 21 24
OD600
hours
Glycerine
ST_LT2 SWE 132V
SWE 152V SWI 30V
SWI 150V
0.0001
0.001
0.01
0.1
1
0 3 6 9 12 15 18 21 24
OD600
hours
Glyecerine+Mannitol
ST_LT2 SWE 132V
SWE 152V SWI 30V
SWI 150V
No difference between
S. Weltevreden and S. Typhimurium LT2
21. • Screening of Carbohydrate Concentration Experiment
Carbohydrate
Concentration
(% -v/v)
Strain
S.
Agona
S.
Seftenberg
S. Typhimurium
LT2
24C 75C 74C 95V 30V 28V
Glucose
0.2 x x x x x x x x x
0.1 x x x x x x x x x
0.05 x x x x x x x x x
0.001 x x x x x x x x x
0.0002 - - - - - - - - -
Mannose
0.2 x x x x x x x x x
0.1 x x x x x x x x x
0.05 x x x x x x x x x
0.001 x x x x x x x x x
0.0002 - - - - - - - - -
Mannitol
0.2 x x x x x x x x x
0.1 x x x x x x x x x
0.05 x x x x x x x x x
0.001 x x x x x x x x x
0.0002 - - - - - - - - -
No difference between
S. Weltevreden and other serovar
x : growth
- : no growth
22. Slide 22
• In vitro cannot verify in silico finding,
might due to:
1. Specific for another not tested
carbon source
2. Have more general function
3. Method that we used is not
appropriate to verify the role of
the system
23. Conclusion
• Our strains:
• Mostly do not harbor AMR gene
• Have the plasmid that common in enterobacteriaceae
family with one of the plasmids (incq1) has relation with
class 1 integron,
• Have point mutation on quinolones at thr57 (57: YS),
• Monophyletic
• Most likely SPI present is 13, 14, and C63PI with some
strains have an additional SPI 3 or 9.
• In a global context geographical relatedness,
possibility of fecal-oral contamination, persistent in
environments
• Phylogenetic structure based on SNP of Vietnam strains have
similarity with the phylogenetic structure of PFGE with the
clonal occurrence convenient characteristic of WGS data to
study the epidemiological or phylogenetic structure
24. Conclusion
• The longer survival of S. Weltevreden in aquatic
environment than other Salmonella serovars might relate
to its specific metabolic activity which absence from
others:
• transport system and
• specific enzyme such as fructose-1,6-bisphosphatase
• We cannot prove the in silico finding with in vitro need
to be worked in the future
25. Future Perspective and Recommendation
• The application of WGS become more common this
study, we use tools from CGE, DTU, Denmark and it shows
the convenient aspect of the tools for commoners that do
not understand bioinformatics
• Data generated from WGS can be used for early detection
of the unwanted system by the presence of certain gene
or mutation, the in-deep study of interested mechanisms,
epidemiology structure study, and many other functions
• Can be used before conducting in vitro assay which is the
advantages as it can reduce the cost for the trial-error
experiment
26. Future Perspective and Recommendation
• The availability of the other WGS data can make it easier
to study the interest microbe in a global context or for
outbreak investigation
• The sequence of the entire genome can detect a small
change that might relate to different phenotyping
expression
• There are several specific systems that have been
identified for S. Weltevreden we cannot test all
hypotheses, therefore, the further experiment to
demonstrate these hypotheses need to be carried out in
the future
• Good understanding better interpretation of metabolic
activity
