Swine microbiota: What’s changing - Dr. Heather Allen, USDA, from the 2012 Allen D. Leman Swine Conference, September 15-18, St. Paul, Minnesota, USA. More presentations at http://www.swinecast.com/2012-leman-swine-conference-material

1. Swine microbiota: what’s changing
Heather K. Allen, PhD
Research Microbiologist
Food Safety and Enteric Pathogens Research Unit

2. Microbial ecology
• “If I could do it all over again, and relive my
vision in the twenty-first century, I would be a
microbial ecologist.”—E. O. Wilson
– 500-1000 bacterial species per mammalian gut
– 10,000,000 virus particles per milliliter of
seawater
• Exploring and hypothesis-testing in complex
environments

3. The Food Safety and Enteric Pathogens
Research Unit
• Reduce foodborne • Alternatives for
pathogen load “growth-promoting”
– Vaccines antibiotics
• E. coli O157:H7
• Salmonella – Define the effects of
• Campylobacter carbadox and ASP250 on
– Pre/probiotics the microbiota
• Inhibitors of Salmonella – Investigate alternatives
growth
– Functional
metagenomics
• For bioactive small
molecules to inhibit
foodborne pathogens

4. Biological Questions
• Is the swine gut
microbiota related to
shedding status?
– What happens to the
swine gut microbiota
during Salmonella
challenge?
• What is the effect of
growth-promoting
antibiotics on the
indigenous microbiota
and phages in swine?

5. Prevalence of Bacterial Foodborne Illness
Pathogen Estimated Annual Mortality
Cases
Salmonella spp 1,027,561 378
Campylobacter spp 845,024 76
Shigella spp 131,254 10
E.coli O157:H7 63,153 20
Listeria spp 1,591 255
Scallan et al. 2011

6. Importance of Salmonella to swine
industry
• 53% of pig farms are positive for Salmonella
• Goal of any strategy (vaccine, feed-additive,
etc.) is to reduce Salmonella carriage
• Assess the swine gut microbiota before and
after Salmonella challenge

7. Experimental design
Inoculated with Salmonella
Non-inoculated
enterica serovar Typhimurium
(NI)
intranasally
6 piglets 54 piglets
0 2 7 21
• enrich for and enumerate Salmonella
• categorize by shedding status
• isolate fecal DNAs
• 16S rRNA gene sequencing

8. Cumulative area under the log curve
Classification of shedders
High shedders
Low shedders
Other shedders HS
LS
0 1 2 7 14 21
Days post inoculation
Bearson et al. submitted Microbes and Infection
Area under the log curve: Huang et al. 2011 PLoS ONE

9. 16S rRNA gene sequence analysis
• Amplified the V1-V3 region using barcoded
primers
• Sequenced on Roche’s 454 Genome
Sequencer on the Titanium platform
• Analyzed in the program mothur
– By operational taxonomic units
(OTUs)
– By closest named relative (phylotypes)

10. Day 0
2
Day 21
7

11. Conclusions from OTU-based analysis
• At day 0, community structure of the “will-be”
LS and HS microbiota was significantly
different
• At day 2, Salmonella-induced changes in the
ecology of the gut caused significant shifts in
the microbiota
• At day 21, community structure of LS and HS
pigs more similar to each other than to NI
pigs, suggesting an impact regardless of
shedding status

12. Who is present?
• Swine microbiota resembles
other mammalian gut
microbiotas
– Dominated by Firmicutes,
Bacteroides, and Proteobacteria
phyla
• Abundance of certain genera is Allen et al. 2011 mBio
uniquely swine
– Relative abundance of Prevotella is
typically over 40%

13. Which bacteria are driving the
shifts at day 2?
Day 2 genera
Statistically
significant
differences:
LS vs. HS
NI vs. HS

14. Day 0

15. Which bacteria were responsible
for the difference at day 0?
All p>0.05
Trends?

16. Next steps
• Systems biology analysis of Salmonella
shedding
Bacterial membership
Swine gene expression data
– Correlations between them
– Shawn Bearson (ARS), Brad Bearson (ARS), Chris
Tuggle (ISU), Jolita Uthe (grad student)

17. Antibiotics used in agriculture are
under scrutiny in the U.S.
• Agricultural antibiotics
have therapeutic and
non-therapeutic
(growth promoting)
uses.
• FDA recently (spring
2012) published a
Guidance For Industry Eckholm, E. “U.S. Meat Farmers Brace
for Limits on Antibiotics”. The New
(#209) to eliminate York Times. 15 September 2010.
growth-promoting
antibiotics in the U.S.

18. The challenges of alternatives to
antibiotics in agriculture
• The mechanism of how antibiotics promote
growth is unclear
– Pathogen prevention or treatment?
– Antinflammatory?
– Decrease pressure of indigenous bacteria on the
immune system?

19. Allen et al., submitted, Trends in Microbiology

20. Biological Questions
• Is the swine gut
microbiota related to
shedding status?
– What happens to the
swine gut microbiota
during Salmonella
challenge?
• What is the effect of
growth-promoting
antibiotics on the
indigenous microbiota
and phages in swine?

21. In-feed antibiotic experiment
Weaning
14 Days Post Farrow
1 week of growth on unamended feed
6 piglets 6 piglets 6 piglets 6 piglets
Control Subtherapeutic Therapeutic ASP250
Age of pigs (in days)
Unamended Unamended Unamended Unamended
21
Day 0 Day 0
Carbadox
35 Unamended 10 g/ton
Day 14 Day 14 Carbadox Day 0
Day 17 (End) 50 g/ton Day 3
Day 42 Day 28 ASP250
77 Unamended
Day 56 Penicillin, chlortetracycline, )
(End Day 0
Day 64 and sulfamethazine Day 8
Day 70 Day 14

22. ASP250 alters bacterial membership
Non-medicated pigs Day 0
ASP250-treated pigs Day 8
Day14
Allen et al. 2011 mBio p<0.01, R=0.43

23. Certain bacterial populations
change significantly with ASP250
• DECREASE: Coprococcus, Succinivibrio,
Streptococcus, Treponema, and Turicibacter
Streptococcus,
spp.
• INCREASE: Escherichia coli
Looft et al., 2012, PNAS

24. E. coli as an indicator of gut
disturbances?
• E. coli populations have been reported to
increase
– with other antibiotics
– In pregnant women
– With diet change in cattle
– In hungry kids in Bangladesh
Looft and Allen, 2012,
Gut Microbes

25. What are the functions of the
community members?
Medicated Non-
• Functions of interest:
(ASP250) medicated
– Mucin degradation
Day 0 Day 0
– Butyrate production
– Antibiotic resistance Day 14 Day 14
• >100 different types of resistance genes in
EACH metagenome
• Swine bacterial metagenomes harbor diverse
antibiotic resistance genes regardless of
antibiotic treatment

26. Gene(s) detected by:
Mechanism of resistance Confer(s) resistance to:
Metagenomics Q-PCR
More prevalent in the treated metagenome
Aminoglycoside O- aph(3′′)-Ib, aph(3′′)-Ib streptomycin Not
phosphotransferase. aph(6′)-Ic, administered
aph(6′)-Id
Class A beta-lactamase. blaTEM-1, beta-lactams
blaSHV-2
Major facilitator superfamily emrD, mdfA, tet(B), bcr chloramphenicol, tetracycline, deoxycholate,
efflux pump mdtH, mdtL, fosfomycin, fosmidomycin, sulfathiazole
rosA, tet(B)
Resistance-nodulation-cell adeA, amrB, acrA fluoramphenicol, aminoglycoside, macrolide,
division efflux pump. mdtF, mdtN, acriflavine, doxorubicin, erythromycin,
mdtO, mdtP, puromycin, beta-lactams
oprA, tolC
Ribosomal protection protein. tet(M) tet(O) tetracycline
More prevalent in the control metagenomes
Resistance-nodulation-cell mexF chloramphenicol, fluoroquinolone
division resistance efflux
pump.
Ribosomal protection protein. tetB(P), tet(Q) tetracycline
Also, more resistance genes in medicated metagenome than non-
medicated (p<0.05)

27. In-feed antibiotic experiment
Weaning
14 Days Post Farrow
1 week of growth on unamended feed
6 piglets 6 piglets 6 piglets 6 piglets
Control Subtherapeutic Therapeutic ASP250
Age of pigs (in days)
Unamended Unamended Unamended Unamended
21
Day 0 Day 0
Carbadox
35 Unamended 10 g/ton
Day 14 Day 14 Carbadox Day 0
Day 17 (End) 50 g/ton Day 3
Day 42 Day 28 ASP250
77 Unamended
Day 56 (End ) Day 0
Day 64 Day 8
Day 70 Day 14

28. Carbadox and gene transfer
• The antibiotic carbadox is
fed to swine to improve
feed efficiency 1. Are other phages
or gene transfer
• VSH-1, a prophage-like
agents induced
element of Brachyspira by carbadox in
hyodysenteriae, is induced the swine gut?
by carbadox 2. Are fitness genes
• Antibiotic resistance genes mobilized?
are transferred by VSH-1
among B. hyo. cells Stanton, T. B. et al. 2008. AEM. 74(10):2950

29. Amplify 16S rRNA genes and
isolate phages
Day 14 15 sampling points
(treatment x time)
Phage extraction
• 16S rRNA gene sequences per individual fecal sample
• 15 phage metagenomes from pooled feces

30. Diverse phages in swine feces
Judi Stasko

31. ASP250 alters phage membership
p<0.1,
Relative abundance
R=0.72

32. Phage integrases are more
abundant with in-feed antibiotics
p<0.01
n=10 n=5
In-feed antibiotics induce prophages in the swine
microbiome

33. Penicillin is likely the component of
ASP250 with phage-related activity
• ASP250 = subinhibitory concentrations of
chlortetracycline, penicillin, sulfamethazine
• PAS = phage-antibiotic synergy
Comeau et al. 2007. PLoS One. 2:e799

34. Conclusions part II
• Increases in E. coli abundance may be a
collateral effect of general ecosystem
disturbances, including antibiotics
• Swine microbial communities harbor diverse
antibiotic resistance genes
• In-feed antibiotics induce prophages in the
swine gut.
• ASP250 causes significant changes in the
membership and abundance of bacterial and
phage communities.

35. Apply microbial ecology to health and
food safety
• Discover targeted approaches to improve food
safety
• Manipulate microbial communities to prevent
carriage of foodborne pathogens
– Use fewer antibiotics
– Identify appropriate alternatives
– Discover novel ways of addressing disease
– Define individual health

36. Acknowledgements
• Salmonella project • Antibiotic alternatives
– Shawn Bearson project
– Brad Bearson – Thad Stanton
– Brian Brunelle – Sam Humphrey
– Jalusa Kich – Stephanie Jones
– Jenn Jones – Michelle Tsai
– Briony Atkinson – Uri Levine
– Torey Looft
• NADC Genomics group
– David Alt, Lea Ann Hobbs, and
Darrell Bayles
• Judi Stasko
• Jim Tiedje and Tim Johnson

37. Analysis of internal controls

38. qPCR for Prevotella and Salmonella