Swine microbiota: what’s changing

Heather K. Allen, PhDResearch Microbiologist

Food Safety and Enteric Pathogens Research Unit

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

The Food Safety and Enteric Pathogens Research Unit

• Reduce foodborne pathogen load

– Vaccines• E. coli O157:H7• Salmonella• Campylobacter

– Pre/probiotics• Inhibitors of Salmonella

growth

– Functional metagenomics• For bioactive small

molecules to inhibit foodborne pathogens

• Alternatives for “growth-promoting” antibiotics

– Define the effects of carbadox and ASP250 on the microbiota

– Investigate alternatives

Biological Questions

• Is the swine gut microbiota related to shedding status?

– What happens to the swine gut microbiotaduring Salmonella challenge?

• What is the effect of growth-promoting antibiotics on the indigenous microbiotaand phages in swine?

Prevalence of Bacterial Foodborne Illness

Pathogen Estimated Annual Cases

Mortality

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

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

Experimental design

Non-inoculated

(NI)

Inoculated with Salmonella

enterica serovar Typhimurium

54 piglets6 piglets

intranasally

0 2 7 21

• enrich for and enumerate Salmonella

• categorize by shedding status

• isolate fecal DNAs

• 16S rRNA gene sequencing

Classification of shedders

0 1 2 7 14 21

Days post inoculationCu

mu

lati

ve a

rea u

nd

er

the

lo

g c

urv

e

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

Bearson et al. submitted Microbes and Infection

High shedders

Low shedders

Other shedders HS

LS

16S rRNA gene sequence analysis

• Amplified the V1-V3 region using barcodedprimers

• 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)

Day 21Day 7Day 2Day 0

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

Who is present?

• Swine microbiota resembles other mammalian gut microbiotas

– Dominated by Firmicutes, Bacteroides, and Proteobacteriaphyla

• Abundance of certain genera is uniquely swine

– Relative abundance of Prevotella is typically over 40%

Allen et al. 2011 mBio

Which bacteria are driving the shifts at day 2?

Day 2 genera

Statistically

significant

differences:

LS vs. HS

NI vs. HS

Day 0

Which bacteria were responsible for the difference at day 0?

All p>0.05

Trends?

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)

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 (#209) to eliminate growth-promoting antibiotics in the U.S.

Eckholm, E. “U.S. Meat Farmers Brace for Limits on Antibiotics”. The New York Times. 15 September 2010.

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?

Allen et al., submitted, Trends in Microbiology

Biological Questions

• Is the swine gut microbiota related to shedding status?

– What happens to the swine gut microbiotaduring Salmonella challenge?

• What is the effect of growth-promoting antibiotics on the indigenous microbiotaand phages in swine?

Age o

f pig

s (

in d

ays)

Weaning

14 Days Post Farrow

6 piglets

Control

6 piglets

Subtherapeutic

Carbadox

10 g/ton

Unamended

Carbadox

50 g/ton

1 week of growth on unamended feed

Day 0

Day 3

Day 28

Day 0

Day 14

Day 17

Day 42

21

35

Day 0

Day 14

Unamended

Unamended

Unamended

6 piglets

Therapeutic

Unamended

(End)

(End )

In-feed antibiotic experiment

6 piglets

ASP250

Day 0

Day 8

Day 14

Day 56

Day 64

Day 70

Unamended

77ASP250

Penicillin, chlortetracycline,

and sulfamethazine

ASP250 alters bacterial membership

Non-medicated pigs

ASP250-treated pigs

Day 0

Day 8

Day14

p<0.01, R=0.43Allen et al. 2011 mBio

Certain bacterial populations change significantly with ASP250

• DECREASE: Coprococcus, Succinivibrio, Streptococcus, Treponema, and Turicibacterspp.

• INCREASE:

Streptococcus,

Escherichia coli

Looft et al., 2012, PNAS

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

What are the functions of the community members?

• Functions of interest:

– Mucin degradation

– Butyrate production

– Antibiotic resistance

• >100 different types of resistance genes in EACH metagenome

• Swine bacterial metagenomes harbor diverse antibiotic resistance genes regardless of antibiotic treatment

Medicated (ASP250)

Non-medicated

Day 0 Day 0

Day 14 Day 14

Mechanism of resistance Gene(s) detected by:

Confer(s) resistance to: Metagenomics Q-PCR

More prevalent in the treated metagenome

Aminoglycoside O-phosphotransferase.

aph(3′′)-Ib, aph(6′)-Ic, aph(6′)-Id

aph(3′′)-Ib streptomycin

Class A beta-lactamase. blaTEM-1, blaSHV-2

beta-lactams

Major facilitator superfamily efflux pump

emrD, mdfA, mdtH, mdtL, rosA, tet(B)

tet(B), bcr chloramphenicol, tetracycline, deoxycholate, fosfomycin, fosmidomycin, sulfathiazole

Resistance-nodulation-cell division efflux pump.

adeA, amrB, mdtF, mdtN, mdtO, mdtP, oprA, tolC

acrA fluoramphenicol, aminoglycoside, macrolide, acriflavine, doxorubicin, erythromycin, puromycin, beta-lactams

Ribosomal protection protein. tet(M) tet(O) tetracycline

More prevalent in the control metagenomes

Resistance-nodulation-cell division resistance efflux pump.

mexF chloramphenicol, fluoroquinolone

Ribosomal protection protein. tetB(P), tet(Q) tetracycline

Not

administered

Also, more resistance genes in medicated metagenome than non-

medicated (p<0.05)

Age o

f pig

s (

in d

ays)

Weaning

14 Days Post Farrow

6 piglets

Control

6 piglets

Subtherapeutic

Carbadox

10 g/ton

Unamended

Carbadox

50 g/ton

1 week of growth on unamended feed

Day 0

Day 3

Day 28

Day 0

Day 14

Day 17

Day 42

21

35

Day 0

Day 14

Unamended

Unamended

Unamended

6 piglets

Therapeutic

Unamended

(End)

(End )

In-feed antibiotic experiment

6 piglets

ASP250

Day 0

Day 8

Day 14

Day 56

Day 64

Day 70

Unamended

77ASP250

Carbadox and gene transfer

• The antibiotic carbadox is fed to swine to improve feed efficiency

• VSH-1, a prophage-like element of Brachyspira hyodysenteriae, is induced by carbadox

• Antibiotic resistance genes are transferred by VSH-1 among B. hyo. cells

Stanton, T. B. et al. 2008. AEM. 74(10):2950

1. Are other phages

or gene transfer

agents induced

by carbadox in

the swine gut?

2. Are fitness genes

mobilized?

Amplify 16S rRNA genes and isolate phages

Day 14

• 16S rRNA gene sequences per individual fecal sample

• 15 phage metagenomes from pooled feces

15 sampling points(treatment x time)

Phage extraction

Diverse phages in swine feces

Judi Stasko

ASP250 alters phage membership

p<0.1,

R=0.72

Rela

tive a

bundance

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

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

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.

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

Acknowledgements

• Salmonella project– Shawn Bearson

– Brad Bearson

– Brian Brunelle

– Jalusa Kich

– Jenn Jones

– Briony Atkinson

• NADC Genomics group– David Alt, Lea Ann Hobbs, and

Darrell Bayles

• Judi Stasko

• Jim Tiedje and Tim Johnson

• Antibiotic alternatives project– Thad Stanton

– Sam Humphrey

– Stephanie Jones

– Michelle Tsai

– Uri Levine

– Torey Looft

Analysis of internal controls

qPCR for Prevotella and Salmonella