Heather K. AllenFood Safety and Enteric Pathogens Research UnitNational Animal Disease Center, Ames, IA

The swine gut microbiota: status and outlook

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/

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

16S rRNA gene sequence analysis to assess the microbiota

www.alimetrics.net

16S rRNA gene

6 Prevotella

4 Clostridium

2 Succinivibrio

4 Roseburia

2 Turicibacter1 Escherichia1 Mitsuokella

Various states of the gut microbiota have been linked to health and disease

Autism

Asthma

Lung infections

Diabetes

Obesity

Gaps in gut microbiota analyses

• Correlation is not causation• Subspecies are often the important ecological

unit

Judi Stasko Jordan Angell

Gaps in gut microbiota analyses

• Correlation is not causation• Subspecies are often the important ecological

unit• Membership does not indicate function

Clostridium

difficileleptum

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?

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)

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 unpublished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

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?

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

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 10countries (half from U. S.)

• Pig age range: pre-wean to slaughter

“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)

Factors impacting the swine gut microbiota*

R-value

GI sampling location 0.53

Study 0.43

Country of origin 0.23Hypervariable 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 Unifracdistance

Gut location is the strongest driver of microbiota differences

Holman et al. BMC Microbiome. submitted

Small intestine

Large intestine

Genera enriched in specific GI samples

Holman et al. BMC Microbiome. submitted

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

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?

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

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.

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

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

Various states of the gut microbiota have been linked to health and disease

http://www.clasado.com/wellness/benefits/gut-microbiota-imbalance/

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

Ruminococcaceae and butyrate

• Ruminococcaceaefamily 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

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

What is the diversity of in swine that shed Salmonella?

but

6 Eubacterium

4 Clostridium

2 Megasphaera

4 Roseburia

2 Butryicoccus1 Faecalibacterium1 Coprococcus

Butyrate transferase gene but

Classification of Salmonella shedders

0 1 2 7 14 21Days post inoculationC

umul

ativ

e ar

ea u

nder

the

log

curv

e

Area under the log curve: Huang et al. 2011 PLoS ONE

High sheddersLow sheddersOther shedders HS

LS

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