dr. heather allen - swine microbiota: what’s changing
DESCRIPTION
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-materialTRANSCRIPT
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