The Swine Gut Microbiota: Status and Outlook - Dr. Heather Allen, National Animal Disease Center, USDA, from the 2016 Allen D. Leman Swine Conference, September 17-20, 2016, St. Paul, Minnesota, USA. More presentations at http://www.swinecast.com/2016-leman-swine-conference-material

1. Heather K. Allen
Food Safety and Enteric Pathogens Research Unit
National Animal Disease Center, Ames, IA
The swine gut
microbiota:
status and
outlook

2. The gut microbiota
• Microbiota = bacterial
community
• 10x more bacterial cells than
human cells in our bodies
• >500 species in a mammalian
gut
http://www.technologyreview.com/news/
418827/you-are-your-bacteria/

3. Measuring the microbiota
• Culture the bacteria
– ~<1% cultivable by any one laboratory technique
• Sequence bacterial DNA (culture-
independent)
– Genomics
– Metagenomics
– 16S rRNA gene sequence analysis
Walker et al 2008
Environmental
Microbiology

4. 16S rRNA gene sequence analysis
to assess the microbiota
www.alimetrics.net
16S rRNA gene
6 Prevotella
4 Clostridium
2 Succinivibrio
4 Roseburia
2 Turicibacter
1 Escherichia
1 Mitsuokella

5. Various states of the gut microbiota have
been linked to health and disease
Autism
Asthma
Lung infections
Diabetes
Obesity

6. Gaps in gut microbiota analyses
• Correlation is not causation
• Subspecies are often the important ecological
unit
Judi Stasko Jordan Angell

7. Gaps in gut microbiota analyses
• Correlation is not causation
• Subspecies are often the important ecological
unit
• Membership does not indicate function
Clostridium
difficileleptum

8. Gaps in gut microbiota analyses
• Correlation is not causation
• Subspecies are often the important ecological
unit
• Membership does not indicate function
• Interactions with the host are complex and
poorly defined
• Is the fecal microbiota representative?
• What are the underlying mechanisms of how
the microbiota influences health and disease?

9. Conclusions from our swine gut
microbiota analyses
• E. coli populations tend to increase with
certain antibiotic treatments (Looft et al. 2012 PNAS; Looft et
al. 2014 ISME J; Looft and Allen 2012 Gut Microbes)
• Ruminococcaceae populations are associated
with decreased Salmonella shedding (Bearson et al.
2013 Infect Genet Evol)
• Firmicutes populations decrease temporarily
with carbadox treatment (Looft et al. 2014 Front Microbiol)

10. Ordination (NMDS) of swine fecal microbiotas
comparing two replicate experiments
Unable to perform direct comparisons
between the years
2014
2013
Trachsel et al.
unpublished
• 100 pigs per study
• Studies conducted 6 months apart

11. Calibrate our analyses of the swine
gut microbiota
• What is the variation in a “healthy” swine gut
microbiota?
• Are certain members always present, i.e., a so-
called core microbiota?
• Does the method of analysis matter?

12. The swine gut microbiota: a meta
analysis
• Analyzed the swine gut microbiota from 20
studies
• Data publically available prior to March 31,
2016
• [Closed-reference picking in QIIME]
Holman et al. BMC Microbiome. submitted

13. The swine gut microbiota: a meta
analysis
• >25 million quality-filtered 16S rRNA gene
sequences from 939 swine GI samples
• Sequences from 3 continents and 10
countries (half from U. S.)
• Pig age range: pre-wean to slaughter

14. “Core” microbiota
• >99% of fecal samples contained the following
bacterial genera:
– Prevotella (an uncultured member was found in
75% of samples!)
– Clostridium
– Alloprevotella
– Ruminococcus
– the RC9 gut group (member of the Rikenellaceae
family, related to Prevotella)

15. Factors impacting the swine gut microbiota*
R-value
GI sampling location 0.53
Study 0.43
Country of origin 0.23
Hypervariable region
sequenced 0.19
Age 0.10
Sequencing platform 0.04
All p<0.001
strongest
weakest
*As
measured
using
analysis of
similarities
(ANOSIM) of
the weighted
Unifrac
distance

16. Gut location is the strongest driver
of microbiota differences
Holman et al. BMC Microbiome. submitted
Small intestine
Large intestine

17. Genera enriched in specific GI
samples
Holman et al. BMC Microbiome. submitted

18. Summary
• Researchers need to publically release their
microbiome data.
• Researchers need to be stewards of their
metadata (i.e., tease apart the “study” effect).
• Gut location is the biggest driver of microbiota
differences—bigger than the study effect.
• A fecal “core” microbiome was tentatively
defined.
– Prevotella, Clostridium, Alloprevotella, Ruminococcus, The RC9 gut group

19. Markers of swine gut microbiota
health…?
• Health is difficult to define
– Even using animal-centric endpoints such as blood
counts, etc., health has a range
– Can analyses of the swine gut microbiota lead to
the definition of a health range for this endpoint?
– Can we manipulate the microbiota toward health?

20. Success in gut microbiota research:
exclusion of Clostridium difficile
• Clostridium difficile, or C. diff., is a serious
intestinal infection causing over 14,000 deaths
annually
• Antibiotic treatment has poor efficacy,
particularly with recurring infections
• Fecal transplant therapy is effective
– In one study of patients with C. diff., resolution of
the infection occurred in 81% of patients receiving
fecal transplantation and only 31% of patients
receiving antibiotics.
www.npr.org

21. Mechanism of efficacy?
• Transplantation of single probiotic bacterium
is not as effective (Reeves et al 2012 Infect Immun; Buffie et al 2015
Nature; ); mixture of different bacteria is important
(Buffie et al 2015 Nature; Lawley et al PLoS Pathogens; Petrof et al. 2013 BMC Microbiome)
• Specific bacterial species, working in concert,
have been identified as protective (Schubert et al. 2015
mBio)
Buffie et al 2015 Nature
• Simplified system—end goal is clear
and measurable
• Other gut microbiota analyses have
murky end goals (“improve health”)
• Microbiota studies that address the
most simple goals will be achieved
first.

22. Outlook for swine gut microbiota
analyses
• The field is progressing from observations to
hypothesis-testing
• Important to conduct network analyses with
host factors
• Need for mechanistic studies of:
– Host-microbe interactions
– Microbiota manipulations
– Bacterial functions

23. Acknowledgements
• Devin Holman
• Brian Brunelle
• Julian Trachsel
• Torey Looft
• Jenn Jones
• Thad Stanton
• Tom Casey
• Animal caretakers
• Mike Marti, Jim Fosse, and Jordan Angell
• Judi Stasko
• Darrell Bayles and David Alt

24. Various states of the gut microbiota have
been linked to health and disease
http://www.clasado.com/wellness/
benefits/gut-microbiota-imbalance/

25. Swine gut microbial ecology
• Swine as model for
humans
• Swine become pork
– Improve swine health
– Improve food safety
• We have defined the
swine gut microbiota
in the small and large
intestine Looft et al. 2014 ISME J

26. Ruminococcaceae and butyrate
• Ruminococcaceae
family members
produce short-chain
fatty acids, including
butyrate
• Butyrate is important
for the maintenance of
the colonic epithelial
barrier and for reduced
intestinal inflammation
Lee and Hase 2014 Nature Chemical Biology

27. Butyrate and Salmonella Shedding
• Calcium-coated butyrate
– Effective at reducing Salmonella colonization in
broilers and pigs
• Dietary Rice Bran
– Dietary fiber fermented to butyrate in distal gut
– Reduced Salmonella colonization in mice

28. What is the diversity of in swine
that shed Salmonella?
but
6 Eubacterium
4 Clostridium
2 Megasphaera
4 Roseburia
2 Butryicoccus
1 Faecalibacterium
1 Coprococcus
Butyrate transferase gene but

29. Classification of Salmonella
shedders
0 1 2 7 14 21
Days post inoculation
Cumulativeareaunderthelogcurve
Area under the log curve: Huang et al. 2011 PLoS ONE
High shedders
Low shedders
Other shedders HS
LS

30. Similarity of the shedders’ butyrate-
producing microbial community
• The diversity of
butyrate-producing
bacteria is different
in swine that
become high
shedders versus low
shedders.
• Manipulate these
results to our
advantage
– Administer butyrate-
producing bacteria
as probiotics