microbiome methods and surrogates - ruminomics · 2016-01-15 · archaeal phylum summaries...
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Ru
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Om
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John Wallace
Microbiome – methods and surrogates
The rumen
The three domains of life
Ruminal microorganisms
50 m
0.5 m
100 m
Ciliate protozoa
106 per g digesta
Anaerobic fungi
103 per g digesta
50 m
Bacteria
1010 per g digesta
Methanogenic archaea
108 per g digesta
2 m
Fermentation
H2 + CO2
CH4
Protozoa, fungi, bacteria
Archaea
Methane production in ruminants
Ru
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Om
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• Community analysis methods
• Proxies for ruminal digesta
• Metaproteomics
• Milk fatty acids vs. methane
Microbiome – methods and surrogates
Secondary structure
of small
subunit ribosomal
RNA
Community analysis based on ss rRNAsequence analysis
Unclassified
Prevotella
Succiniclasticum
Acidaminococcus
Dialister
Butyrivibrio
Ruminococcus
Treponema
Syntrophococcus
Fibrobacter
Selenomonas
Acetitomaculum
Incertae Sedis
Pseudobutyrivibrio
Atopobium
Mogibacterium
Community analysis based on ss rRNAsequence analysis
Ru
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Om
icsRuminOmics ring test
• Seven international labs
• DNA from six rumen samples
distributed
• “Just tell us what you think is there”
RuminOmics ring testBacterial phylum summaries
Participating laboratories
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Teagasc AgResearch CNRS CSIRO ARK
Thermoplasmatales
Methanosarcinales
Methanomicrobiales
Methanobacteriales
F B D E F
RuminOmics ring testArchaeal phylum summaries
Participating laboratories
RuminOmics ring testReaction
“bury it”
RuminOmics ring testOngoing investigation on reasons for
differences
• Choice of hypervariable region
• Sequencing platform
• Taxonomic software
• Databases
• What is the correct answer???
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Buccal-ruminal-faecal microbiomes
RuminOmics surrogates study
Proxies for rumen sampling – mouth (swab and bolus) and faeces
Proxies for rumen sampling – mouth (swab and bolus) and faeces
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Metaproteomics: 2-D gel electrophoresis, different samples
Metaproteomics: 2-D gel electrophoresis, same sample different gels
Metaproteomics: 2-D gel electrophoresis, different samples
Archaealenzymes
Protozoal actins
Bacteroidetes
Firmicutes
Proteobacteria
Archaea
Protists
Fungi
Metazoa
Results of 2-D gel electrophoresis: phylogenetic analysis
• Ultimate 3000 RS LC nano system
• LQT Orbitrap Velos/Q Exactive
• Proteome Discoverer software
Shotgun metaproteomics
UniProt• Taxonomy
• Function• Gene Ontology (GO terms)
Results of shotgun metaproteomics: phylogenetics and ontology
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Can milk fatty acids predict methane emissions?
• 1000 cows
• 195 fatty acids in milk
• Four countries
• Seven farms
0.2 0.4 0.6 0.8 1.0 1.2
10
15
20
25
30
35
40
c12-18:1
c12-18:1
CH
4g
.kg
.DM
I
BianchiniCAMBS
FranciosiGandolfiMinkiö
NOTTSRöbäcksdalen
Typical results for a single fatty acid in milk
All FA with significant positive within farm correlations
iso13:0 anteiso13:0 iso14:0 iso15:0 iso16:0
16:0 iso18:0 18:0 20:0 22:0
24:0 c15-24:1 Unid 16 SFA
All FA with significant negative within farm correlations
5:0 7:0 9:0 11:0 13:0 c9-13:1 14:0
c9-14:1 15:0 t8-16:1 t13-16:1 c10-16:1
t9,t13-16:2 + t10-17:1 c9,c12-16:2 c10-17:1
t6-8 18:1 t9-18:1 t10-18:1 t12-18:1 t15-18:1 c11-18:1
c12-18:1 c13-18:1 unid 9 c13-18:1 + unid 9 t16-18:1 + c14-18:1
c15-18:1 c16-18:1 sum trans 18:1 t9,t13-18:2
19:0 t10,t14-18:2 19:0 + t10,t14-18:2 t9,t14-18:2 t9,t12-18:2
c9,t13-18:2 + c10,t14-18:2 c6,c12-18:2 c9,t14-18:2 c9,t12-18:2
t9,c12-18:2 c9,c12-18:2 (+ c9,c15-18:2) c12,c15-18:2 trans 18:2
t12,t14-CLA + t12,c14-CLA t11,t13-CLA t9,t11-CLA + 18:4n-3
t10,c12-CLA + c11,t13-CLA + c15-20:1 t9,c11-CLA sum CLA 18:3(n-6)
c7-19:1 c10-19:1 c11-19:1 c10-19:1 + c11-19:1 sum cis 19:1
t13-20:1 c13-20:1 20:2(n-6) + unid 14 20:3(n-6) Unid 1 Unid 3 Unid 6 Unid 24 Trans fatty acids PUFA c9-20:1/(c9-20:1+20:0)
All FA with significant positive within farm correlations
iso13:0 anteiso13:0 iso14:0 iso15:0 iso16:0
16:0 iso18:0 18:0 20:0 22:0
24:0 c15-24:1 Unid 16 SFA
All FA with significant negative within farm correlations
5:0 7:0 9:0 11:0 13:0 c9-13:1 14:0
c9-14:1 15:0 t8-16:1 t13-16:1 c10-16:1
t9,t13-16:2 + t10-17:1 c9,c12-16:2 c10-17:1
t6-8 18:1 t9-18:1 t10-18:1 t12-18:1 t15-18:1 c11-18:1
c12-18:1 c13-18:1 unid 9 c13-18:1 + unid 9 t16-18:1 + c14-18:1
c15-18:1 c16-18:1 sum trans 18:1 t9,t13-18:2
19:0 t10,t14-18:2 19:0 + t10,t14-18:2 t9,t14-18:2 t9,t12-18:2
c9,t13-18:2 + c10,t14-18:2 c6,c12-18:2 c9,t14-18:2 c9,t12-18:2
t9,c12-18:2 c9,c12-18:2 (+ c9,c15-18:2) c12,c15-18:2 trans 18:2
t12,t14-CLA + t12,c14-CLA t11,t13-CLA t9,t11-CLA + 18:4n-3
t10,c12-CLA + c11,t13-CLA + c15-20:1 t9,c11-CLA sum CLA 18:3(n-6)
c7-19:1 c10-19:1 c11-19:1 c10-19:1 + c11-19:1 sum cis 19:1
t13-20:1 c13-20:1 20:2(n-6) + unid 14 20:3(n-6) Unid 1 Unid 3 Unid 6 Unid 24 Trans fatty acids PUFA c9-20:1/(c9-20:1+20:0)
Methane α Fatty acid saturation• Reflects H2 availability for
both
All FA with significant positive within farm correlations
iso13:0 anteiso13:0 iso14:0 iso15:0 iso16:0
16:0 iso18:0 18:0 20:0 22:0
24:0 c15-24:1 Unid 16 SFA
All FA with significant negative within farm correlations
5:0 7:0 9:0 11:0 13:0 c9-13:1 14:0
c9-14:1 15:0 t8-16:1 t13-16:1 c10-16:1
t9,t13-16:2 + t10-17:1 c9,c12-16:2 c10-17:1
t6-8 18:1 t9-18:1 t10-18:1 t12-18:1 t15-18:1 c11-18:1
c12-18:1 c13-18:1 unid 9 c13-18:1 + unid 9 t16-18:1 + c14-18:1
c15-18:1 c16-18:1 sum trans 18:1 t9,t13-18:2
19:0 t10,t14-18:2 19:0 + t10,t14-18:2 t9,t14-18:2 t9,t12-18:2
c9,t13-18:2 + c10,t14-18:2 c6,c12-18:2 c9,t14-18:2 c9,t12-18:2
t9,c12-18:2 c9,c12-18:2 (+ c9,c15-18:2) c12,c15-18:2 trans 18:2
t12,t14-CLA + t12,c14-CLA t11,t13-CLA t9,t11-CLA + 18:4n-3
t10,c12-CLA + c11,t13-CLA + c15-20:1 t9,c11-CLA sum CLA 18:3(n-6)
c7-19:1 c10-19:1 c11-19:1 c10-19:1 + c11-19:1 sum cis 19:1
t13-20:1 c13-20:1 20:2(n-6) + unid 14 20:3(n-6) Unid 1 Unid 3 Unid 6 Unid 24 Trans fatty acids PUFA c9-20:1/(c9-20:1+20:0)
Methane α (1/Odd number FAs) • Reflects fatty acid synthesis
from propionate• Propionate ↓ as CH4↑
All FA with significant positive within farm correlations
iso13:0 anteiso13:0 iso14:0 iso15:0 iso16:0
16:0 iso18:0 18:0 20:0 22:0
24:0 c15-24:1 Unid 16 SFA
All FA with significant negative within farm correlations
5:0 7:0 9:0 11:0 13:0 c9-13:1 14:0
c9-14:1 15:0 t8-16:1 t13-16:1 c10-16:1
t9,t13-16:2 + t10-17:1 c9,c12-16:2 c10-17:1
t6-8 18:1 t9-18:1 t10-18:1 t12-18:1 t15-18:1 c11-18:1
c12-18:1 c13-18:1 unid 9 c13-18:1 + unid 9 t16-18:1 + c14-18:1
c15-18:1 c16-18:1 sum trans 18:1 t9,t13-18:2
19:0 t10,t14-18:2 19:0 + t10,t14-18:2 t9,t14-18:2 t9,t12-18:2
c9,t13-18:2 + c10,t14-18:2 c6,c12-18:2 c9,t14-18:2 c9,t12-18:2
t9,c12-18:2 c9,c12-18:2 (+ c9,c15-18:2) c12,c15-18:2 trans 18:2
t12,t14-CLA + t12,c14-CLA t11,t13-CLA t9,t11-CLA + 18:4n-3
t10,c12-CLA + c11,t13-CLA + c15-20:1 t9,c11-CLA sum CLA 18:3(n-6)
c7-19:1 c10-19:1 c11-19:1 c10-19:1 + c11-19:1 sum cis 19:1
t13-20:1 c13-20:1 20:2(n-6) + unid 14 20:3(n-6) Unid 1 Unid 3 Unid 6 Unid 24 Trans fatty acids PUFA c9-20:1/(c9-20:1+20:0)
Methane α Branched FAs • Reflects fatty acid synthesis
from short-chain branched VFA
Predominant substrate (from Hobson and Stewart, 1997), fermentation end products (from Russell and Rychlik, 2001) and OBCFA profile (g/100 g fatty acids; original references in Vlaeminck et al., 2006a) of
some major bacteria involved in rumen carbohydrate fermentation. Predominant OBCFA is underlined. Indication of main substrate is given by superscript letter.
R. albusa
Fermentation productsd
A
Anteiso C13:0
–
Anteiso C15:0
9.4
Anteiso C17:0
1.3
Iso C13:0
–
Iso C15:0
–
Iso C17:0
0.7
Iso C14:0
20.6
Iso C16:0
11.0
C13:0
–
C15:0
10.3
C17:0
1.4
C17:1
–
B. fibrisolvensa A, B, F 6.4 16.2 8.6 6.8 10.4 5.7 10.8 11.1 2.9 7.8 4.3 3.5
R. flavefaciensa
S. amylolyticab
A, S
A, P
– 2.3 2.9 – 35.7 5.2 2.5 7.3 0.1 3.2 0.5 –
N6 – – – – 52.6 10.8 1.6 5.3 1.6 5.0 – –
B24 – – – – 0.1 0.3 – 0.6 1.4 3.3 1.3 0.6
Prevotellab , c A, S 1.2 36.7 4.2 3.0 14.7 2.3 3.3 3.0 1.2 12.1 2.1 –
L. multiparusb , c A, L, F – 4.0 2.6 – 1.1 1.1 1.2 1.8 0.3 2.9 0.8 0.1
S. dextrinosolvensc A, S 0.8 3.6 1.0 – 0.1 – 0.6 1.5 0.5 4.0 0.7 –
R. amylophilusb A, S, F – 1.1 – – – – – – 0.5 1.1 0.3 0.1
F. succinogenesa A, S 3.9 7.7 1.2 – 0.1 0.2 3.6 3.4 9.0 30.2 2.1 –
S. bovisb L – 0.9 – – – – 0.4 0.2 0.6 1.7 1.2 0.2
M. elsdeniic
E. ruminantiumb
A, P, B
B, L, F
– 2.8 – 0.1 0.2 0.2 1.5 0.5 1.5 6.0 4.5 3.0
B1C23 – – – – 17.7 1.4 – – 5.4 49.0 1.5 –
GA195 – 30.1 1.7 – 0.4 0.2 6.1 3.7 0.4 6.5 0.4 –
S. ruminantiumb A, P, L – 0.1 – – 0.2 – 0.3 0.1 1.3 6.0 2.9 2.6
a Bacteria fermenting cellulose and hemicellulose.
b Bacteria fermenting starch.
c Bacteria fermenting sugar and pectin.
d A: acetate; S: succinate; B: butyrate; F formate; P: propionate; L: lactate.
Different ruminal bacteria contain different minor fatty acids
Fievez et al. (2012)
All FA with significant positive within farm correlations
iso13:0 anteiso13:0 iso14:0 iso15:0 iso16:0
16:0 iso18:0 18:0 20:0 22:0
24:0 c15-24:1 Unid 16 SFA
All FA with significant negative within farm correlations
5:0 7:0 9:0 11:0 13:0 c9-13:1 14:0
c9-14:1 15:0 t8-16:1 t13-16:1 c10-16:1
t9,t13-16:2 + t10-17:1 c9,c12-16:2 c10-17:1
t6-8 18:1 t9-18:1 t10-18:1 t12-18:1 t15-18:1 c11-18:1
c12-18:1 c13-18:1 unid 9 c13-18:1 + unid 9 t16-18:1 + c14-18:1
c15-18:1 c16-18:1 sum trans 18:1 t9,t13-18:2
19:0 t10,t14-18:2 19:0 + t10,t14-18:2 t9,t14-18:2 t9,t12-18:2
c9,t13-18:2 + c10,t14-18:2 c6,c12-18:2 c9,t14-18:2 c9,t12-18:2
t9,c12-18:2 c9,c12-18:2 (+ c9,c15-18:2) c12,c15-18:2 trans 18:2
t12,t14-CLA + t12,c14-CLA t11,t13-CLA t9,t11-CLA + 18:4n-3
t10,c12-CLA + c11,t13-CLA + c15-20:1 t9,c11-CLA sum CLA 18:3(n-6)
c7-19:1 c10-19:1 c11-19:1 c10-19:1 + c11-19:1 sum cis 19:1
t13-20:1 c13-20:1 20:2(n-6) + unid 14 20:3(n-6) Unid 1 Unid 3 Unid 6 Unid 24 Trans fatty acids PUFA c9-20:1/(c9-20:1+20:0)
Ruminococcus albus - H2 producer Ruminococcus flavefaciens - H2
producer
Fibrobacter succinogenes – non-H2 producer
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Conclusions
• (Unspoken) problems of
community analysis are being
resolved
• Oral samples are useful proxies
for ruminal community analysis
• Metaproteomics interesting for
microbiome, but practical
usefulness is limited
• Milk fatty acids vs. methane
makes sense, but predictive
value between farms is limited
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