Linda van der Graaf van Bloois -...

Whole genome analysis of Campylobacter fetus

indicates recent diversification

and

reveals inconsistency of phenotypic and genomic characteristics

Linda van der Graaf – van Bloois

Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, WHO Collaborating Centre for Campylobacter / OIE Reference Laboratory for

Campylobacteriosis

Rapid Microbial NGS and Bioinformatics: Translation Into Practice, 2016

Campylobacter fetus subspecies

Pathogen in both animals and humans

- C. fetus subsp. venerealis (Cfv) and biovar intermedius (Cfvi)

restricted to the genital tract of both male and female cattle

causative agent of Bovine Gential Campylobacteriosis

- C. fetus subsp. fetus (Cff)

isolated from a variety of sites and different hosts

- C. fetus subsp. testudinum (Cft)

isolated from reptiles and humans with underlying illness

Background

Phenotypic subspecies differentiation

1% Glycine tolerance (growth in presence of 1% glycine)

H2S production in cysteine-rich medium

1% Glycine Tolerance H2S production

Cff + +

Cfv - -

Cfvi - +

Background

Molecular characterisation

MLST ST sap type Core genome

(1509 ORFs)

Cfv 4 A A

Cfvi 4 A A

Cff variable A and B B

Background

Aims

- Genome-wide SNP-based phylogenetic analysis

- Specific SNPs or genes that could be associated with the

phenotypes of the C. fetus strains

- BEAST analysis to estimate the divergence dates of Cff and Cfv

Whole Genome Sequencing

- 41 C. fetus strains from different countries and sources

- Roche 454 and Illumina MiSeq sequencing of 22 C. fetus strains

3 genomes closed with PacBio

- Illumina Miseq sequencing of 13 strains

- NCBI genbank genomes

2 Cff strains

3 Cfv strains

1 Cfvi strain

Methods

Whole Genome Analysis

- Genome alignment – Parsnp

- Recombination regions – Gubbins

- Comparison of SNPs and genes with phenotypes

- BEAST – recombination filtered non-synonymous SNPs from Gubbins

results

- Calculation of branch-specific dN/dS ratios

Methods

Phylogenetic analysis SNPs core genomes

Results

Genotype

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFVI

CFVI

CFVI

CFVI

CFV

CFV

CFV

CFV

CFV

CFV

CFV

CFVI

CFVI

CFVI

CFVI

CFVI

CFVI

CFVI

CFVI

CFVI

CFVI

CFVI

1. Sap B, ST 3

4. Sap A, ST 4 and 6

3. Sap A, ST 2

5. Sap A, ST 4

2. Sap B, ST 5

Clade-specific core genome SNPs

Results

Genotype

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFF

CFVI

CFVI

CFVI

CFVI

CFV

CFV

CFV

CFV

CFV

CFV

CFV

CFVI

CFVI

CFVI

CFVI

CFVI

CFVI

CFVI

CFVI

CFVI

CFVI

CFVI

1. Sap B, ST 3


3. Sap A, ST 2

5. Sap A, ST 4

2. Sap B, ST 5

Core genome SNPs specific for C. fetus clades

Clade Representing Number

of clade-

specific

SNPs

Number of clade-specific orthologs (annotation)

1 Cff serotype B 1547 2 (hypothetical)

2 Cff serotype B 1703 41 (fic, transposase, hypothetical)

3 Cff serotype A 1430 6 (R-M system type I and III)

4 Cff serotype A 121 0

5 Cfv and Cfvi 282 0

5 Cfv 5 6

1-2 Cff serotype B, total 571 14 (toxin/antitoxin, R-M, S-layer associated)

3-4 Cff serotype A, total 83 14 (glycosyltransferase, methyltransferase, ketoreductase)

Core genomes clusters vs phenotypes

Results

1. Sap B, ST 3


3. Sap A, ST 2

5. Sap A, ST 4

2. Sap B, ST 5

Genotype Phenotype

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF n.a.

CFF CFF

CFF CFF

CFVI CFVI

CFVI CFVI

CFVI n.a.

CFVI CFVI

CFV CFV

CFV CFV

CFV n.a.

CFV CFV

CFV CFV

CFV CFV

CFV CFV

CFVI CFVI

CFVI CFF

CFVI CFVI

CFVI CFVI

CFVI CFF

CFVI CFVI

CFVI CFVI

CFVI CFVI

CFVI CFF

CFVI CFV

CFVI n.a.

Recombination regions

Results

1. Sap B, ST 3


3. Sap A, ST 2

5. Sap A, ST 4

2. Sap B, ST 5

Genotype Phenotype

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF n.a.

CFF CFF

CFF CFF

CFVI CFVI

CFVI CFVI

CFVI n.a.

CFVI CFVI

CFV CFV

CFV CFV

CFV n.a.

CFV CFV

CFV CFV

CFV CFV

CFV CFV

CFVI CFVI

CFVI CFF

CFVI CFVI

CFVI CFVI

CFVI CFF

CFVI CFVI

CFVI CFVI

CFVI CFVI

CFVI CFF

CFVI CFV

CFVI n.a. S-layer associated

Sugar biosynthesis and CjeI

flgE2

unique

shared recombination events

Aims


- Specific SNPs or genes that could be

associated with the phenotypes of the C.

fetus strains

- BEAST analysis to estimate the divergence dates of Cff and Cfv

Putative cysteine transporter

Results

Cff 82-40

Cfv 97/608

H2S production vs cysteine transporter

Results

1. Sap B, ST 3


3. Sap A, ST 2

5. Sap A, ST 4

2. Sap B, ST 5

Genotype Phenotype H2S production Cysteine transporter CFF CFF + +

CFF CFF + +

CFF CFF + +

CFF CFF + +

CFF CFF + +

CFF CFF + +

CFF CFF + +

CFF CFF + +

CFF CFF + +

CFF CFF + +

CFF CFF + +

CFF CFF + +

CFF CFF + +

CFF CFF + +

CFF CFF + +

CFF CFF + +

CFF n.a. n.a. +

CFF CFF + +

CFF CFF + +

CFVI CFVI + +

CFVI CFVI + +

CFVI n.a. n.a. +

CFVI CFVI + +

CFV CFV - -

CFV CFV - -

CFV n.a. n.a. -

CFV CFV - -

CFV CFV - -

CFV CFV - -

CFV CFV - -

CFVI CFVI + +

CFVI CFF + +

CFVI CFVI + +

CFVI CFVI + +

CFVI CFF + +

CFVI CFVI + +

CFVI CFVI + +

CFVI CFVI + +

CFVI CFF + +

CFVI CFV - -

CFVI n.a. n.a. +

Aims


- Specific SNPs or genes that could be associated with the

phenotypes of the C. fetus strains

- BEAST analysis to estimate the divergence

dates of Cff and Cfv

BEAST analysis

Results

- Recombination filtered non-synonymous SNPs from Gubbins - Reptile-associated C. fetus testudinum as outgroup

BEAST analysis

Results

BEAST analysis

Results

1. Sap B, ST 3

2. Sap B, ST 5

3. Sap A, ST 2

4. Sap A, ST 4,6

5. Sap A, ST 4

dN/dS ratio’s

Results

dN/dS: non-synonymous / synonymous SNPs

1. Sap B, ST 3

2. Sap B, ST 5

3. Sap A, ST 2

4. Sap A, ST 4,6

5. Sap A, ST 4

Conclusions

- Genome-wide SNP analysis divided Cff and Cfv strains in 5 clades

- Clades were inconsistent with phenotypes of strains

Phenotype

- 1% glycine tolerance; unknown

- H2S production: Partial deletion of putative cysteine transporter is

observed in H2S negative strains (Cfv)

- BEAST showed that Cfv strains evolved recently from Cff ancestor

under diversifying selection

Discussion

- Phenotype used for differentiation Cff and Cfv in BGC control programs

- Phenotype is inconsistent with genotype

- Significance phenotypic identification?

- Cluster 5 with Cfv/Cfvi is probably successful bovine clone

Core genomes clusters vs phenotypes

Results

1. Sap B, ST 3


3. Sap A, ST 2

5. Sap A, ST 4

2. Sap B, ST 5

Genotype Phenotype

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF CFF

CFF n.a.

CFF CFF

CFF CFF

CFVI CFVI

CFVI CFVI

CFVI n.a.

CFVI CFVI

CFV CFV

CFV CFV

CFV n.a.

CFV CFV

CFV CFV

CFV CFV

CFV CFV

CFVI CFVI

CFVI CFF

CFVI CFVI

CFVI CFVI

CFVI CFF

CFVI CFVI

CFVI CFVI

CFVI CFVI

CFVI CFF

CFVI CFV

CFVI n.a.

Acknowledgments

Aldert Zomer

Birgitta Duim

Jaap Wagenaar

Bill Miller and Emma Yee, USDA, Albany USA

Ken Forbes, University of Aberdeen, UK

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