Dr. Heather Allen - Swine microbiota: What’s changing

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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

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  • When I was a rotating graduate student, I was grabbed by the same fascination that Dr. Wilson beholds when I spent 6 weeks in Jo Handelsman’s lab. Nothing in my undergraduate education captivated me as thoroughly as microbiology.
  • Plug Vijay
  • Metagenomics gives us the power to ask this question. Idea would be to identify factors that the antibiotics alter to promote growth. Then develop novel approaches that have the same growth-promoting effect without increasing the prevalence of antibiotic resistance.
  • Animals are often asymptomatic carriers of Salmonella, serving as a reservoir for the foodborne pathogenSalmonellais present in the environment so the goal of eliminating Salmonella is a bit far-fetched. But if it can be reduced, then the number of illnesses are reduced, and that is a good thing.
  • I know that not all of you are microbiologists, but given the attention that the HMP is getting and that some of you are microbiologists I thought I’d give a general informational slide about the swine microbiota.
  • Chlortetracycline and carbadox make up almost 75% of antibiotic use in feed of nursery age pigs
  • Metagenomics gives us the power to ask this question. Idea would be to identify factors that the antibiotics alter to promote growth. Then develop novel approaches that have the same growth-promoting effect without increasing the prevalence of antibiotic resistance.
  • Lower LAB in folks with higher BMIFumaric acid mirrors the effects of ASP250 in regards to the E. coli and lactobacilli
  • 1) sub-conc of penicillin weakened streptococcus such that phage resistant strains in mixed cultures were susceptible to phage lysis by exogenous phages (Verhue 1978)2) PAS Aztreonam and cefiximeNot tetracycline orgentamycin
  • Dr. Heather Allen - Swine microbiota: What’s changing

    1. 1. Swine microbiota: what’s changing Heather K. Allen, PhD Research Microbiologist Food Safety and Enteric Pathogens Research Unit
    2. 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. 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. 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. 5. Prevalence of Bacterial Foodborne Illness Pathogen Estimated Annual Mortality CasesSalmonella spp 1,027,561 378Campylobacter spp 845,024 76Shigella spp 131,254 10E.coli O157:H7 63,153 20Listeria spp 1,591 255 Scallan et al. 2011
    6. 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. 7. Experimental design Inoculated with SalmonellaNon-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. 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 InfectionArea under the log curve: Huang et al. 2011 PLoS ONE
    9. 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. 10. Day 0 2Day 21 7
    11. 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. 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. 13. Which bacteria are driving the shifts at day 2?Day 2 generaStatisticallysignificantdifferences:LS vs. HSNI vs. HS
    14. 14. Day 0
    15. 15. Which bacteria were responsible for the difference at day 0? All p>0.05 Trends?
    16. 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. 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. 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. 19. Allen et al., submitted, Trends in Microbiology
    20. 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. 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 ASP250Age 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. 22. ASP250 alters bacterial membership Non-medicated pigs Day 0 ASP250-treated pigs Day 8 Day14Allen et al. 2011 mBio p<0.01, R=0.43
    23. 23. Certain bacterial populations change significantly with ASP250 • DECREASE: Coprococcus, Succinivibrio, Streptococcus, Treponema, and Turicibacter Streptococcus, spp. • INCREASE: Escherichia coliLooft et al., 2012, PNAS
    24. 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. 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. 26. Gene(s) detected by: Mechanism of resistance Confer(s) resistance to: Metagenomics Q-PCRMore prevalent in the treated metagenomeAminoglycoside O- aph(3′′)-Ib, aph(3′′)-Ib streptomycin Notphosphotransferase. aph(6′)-Ic, administered aph(6′)-IdClass A beta-lactamase. blaTEM-1, beta-lactams blaSHV-2Major 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, tolCRibosomal protection protein. tet(M) tet(O) tetracyclineMore prevalent in the control metagenomesResistance-nodulation-cell mexF chloramphenicol, fluoroquinolonedivision resistance effluxpump.Ribosomal protection protein. tetB(P), tet(Q) tetracycline Also, more resistance genes in medicated metagenome than non- medicated (p<0.05)
    27. 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 ASP250Age 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. 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. 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. 30. Diverse phages in swine feces Judi Stasko
    31. 31. ASP250 alters phage membership p<0.1, Relative abundance R=0.72
    32. 32. Phage integrases are moreabundant with in-feed antibiotics p<0.01 n=10 n=5In-feed antibiotics induce prophages in the swine microbiome
    33. 33. Penicillin is likely the component ofASP250 with phage-related activity• ASP250 = subinhibitory concentrations of chlortetracycline, penicillin, sulfamethazine• PAS = phage-antibiotic synergy Comeau et al. 2007. PLoS One. 2:e799
    34. 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. 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. 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. 37. Analysis of internal controls
    38. 38. qPCR for Prevotella and Salmonella

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