Mammalian Microbiomes CONFIDENCE O.JDSPCJPMPHJDBMEFOUJGJDBUJPOGSPN$IBSMFT3JWFS"VTUSBMJBBOE/FX;FBMBOE

OFFICIALOFFICIAL JOURNAL JOURNAL OF OF THE THE AUSTRALIAN AUSTRALIAN SOCIETY SOCIETY FOR FOR MICROBIOLOGY MICROBIOLOGY INC. INCINC.

VolumeVolume 3636 NumberNumber 11 MarchMarch 20152015

Mammalian microbiomes CONFIDENCE *O.JDSPCJPMPHJDBM*EFOUJGJDBUJPOGSPN$IBSMFT3JWFS"VTUSBMJBBOE/FX;FBMBOE

At Charles River, we know that the safety of your manufacturing environment depends on a clear picture of your microbial risk. Our solution? Accugenix®. Unmatched for its relevance and coverage, our microbial database paired with sequencing and MALDI-TOF provide an unparalleled 98% accuracy rate. Together with our tracking and trending tools, we deliver the precision and data yoV need to monitor and report your state of control with confidence.

Learn more about our Accugenix® solutions at www.criver.com/accugenix.

"1"44*0/'03*//07"5*0/ Building C, Suite G04, Rodborough Road 'PSFORVJSJFTDPOUBDU [email protected] Frenchs Forest, NSW, 2086 (02)94547701 The Australian Society for Microbiology Inc. OFFICIAL JOURNAL OF THE AUSTRALIAN SOCIETY FOR MICROBIOLOGY INC. 9/397 Smith Street Fitzroy, Vic. 3065 Tel: 1300 656 423 Volume 36 Number 1 March 2015 Fax: 03 9329 1777 Email: [email protected] www.theasm.org.au Contents ABN 24 065 463 274 Vertical For Microbiology Australia Transmission 2 correspondence, see address below. Jon Iredell Editorial team Prof. Ian Macreadie, Mrs Jo Macreadie Guest Editorial 3 and Mrs Hayley Macreadie Mammalian microbiomes 3 Editorial Board Linda L Blackall Dr Chris Burke (Chair) Dr Gary Lum Prof. Mary Barton Dr John Merlino In Focus 4 Prof. Linda Blackall Prof. Wieland Meyer Prof. Sharon Chen Prof. William Rawlinson Methane matters in animals and man: from beginning to end 4 Prof. Peter Coloe Dr Paul Selleck Emily Hoedt, Paul Evans, Stuart Denman, Chris McSweeney, Dr Narelle Fegan Dr David Smith Dr Geoff Hogg Ms Helen Smith Paraic ÓCuív and Mark Morrison Prof.. Jonathan Iredell Dr Jack Wang Dr Ipek Kurtböke Dr Paul Young The marine mammal microbiome: current knowledge and future directions 8 Subscription rates Current subscription rates are available Tiffanie M Nelson, Amy Apprill, Janet Mann, from the ASM Melbourne ofﬁ ce. Tracey L Rogers and Mark V Brown Editorial correspondence The role of the gut microbiome in host systems 14 Prof. Ian Macreadie/Mrs Jo Macreadie Tel: 0402 564 308 (Ian) Clarissa Febinia, Connie Ha, Chau Le and Andrew Holmes Email: [email protected] Under the Published four times a year Microscope 18 in print and open access online by Modulation of the rumen microbiome 18 Rosalind Gilbert, Diane Ouwerkerk and Athol Klieve Polymicrobial nature of chronic oral disease 22 Stuart Dashper, Helen Mitchell, Geoff Adams and Eric Reynolds Unipark, Building 1, Level 1 195 Wellington Road, Clayton, Vic. 3168 Gastrointestinal microbiota, diet and brain functioning 25 http://microbiology.publish.csiro.au Shakuntla Gondalia and Andrew Scholey Publishing enquiries Marsupial oral cavity microbiome 29 Jenny Bennett Email: [email protected] Philip S Bird, Wayne SJ Boardman, Darren J Trott and Linda L Blackall Production enquiries Lab Helen Pavlatos Report 32 Email: [email protected] Relative abundance of Mycobacterium in ovine Johne’s disease 32 Advertising enquiries Andy O Leu, Paul Pavli, David M Gordon, Jeff Cave, Jacek M Gowzdz, Doug Walters Nick Linden, Grant Rawlin, Gwen E Allison and Claire L O’Brien Tel: 03 9545 8505 Mobile: 0419 357 779 ASM Email: [email protected] Affairs 37 © 2015 The Australian Society for Microbiology Inc. Interactions with other microbiology societies The ASM, through CSIRO Publishing, reserve all rights to the content, artwork and photographs in Microbiology through Microbiology Australia 37 Australia. Permission to reproduce text, photos and artwork must be sought from CSIRO Publishing. ASM History SIG: Microbiology Australia 37 The Australian Copyright Act 1968 and subsequent amendments permit downloading and use of an article by Recent developments in virology by Australian researchers 38 an individual or educational institution for non- commercial personal use or study. Multiple reproduction Clinical Serology and Molecular SIG 39 of any Microbiology Australia article in a study block is governed by rights agreement managed by Copyright Report from the ASM Antimicrobial Special Interest Group (ASIG) 40 Agency Limited and fees may apply. Authors published in Microbiology Australia have the moral right under Australian law to be acknowledged as the creator. ISSN 1324-4272 eISSN 2201-9189 While reasonable effort has been made to ensure the accuracy of the content, the Australian Society for Microbiology, CSIRO, and CSIRO Publishing accept no responsibility for any loss or damage from the direct or indirect use of or reliance on the content. The opinions expressed in articles, letters, and advertisements in Microbiology Australia are not necessarily those of the Australian Society for Microbiology, the Editorial Board, CSIRO, and CSIRO Publishing. Cover image: Background is faecal homogenate from mouse that has been Gram stained. Photo credit from Yi Vee Chew and Andy Holmes (University of Sydney).

MICROBIOLOGY AUSTRALIA • MARCH 2015 1 Vertical Transmission

regarded as conventional or traditional microbiology. We can look to the annual scientiﬁc meeting as a venue for transdisciplinary microbiology that is difﬁcult to manage in highly specialised meetings. This shift will be seen at the integrated symposia in Canberra (ASM 2015), in the themed meeting in Perth (ASM 2016) and in the planning of ASM 2017 in Hobart.

Jon Iredell A link to state branches and the Visiting Speaker program and other President of ASM well-established infrastructure is another easy route that ASM provides for members to work through new proposals, and give early career researchers and professional microbiologists a taste of con-

Along with all the sciences, our discipline is evolving quickly in all ference organising and a chance to test ideas. This is managed easily areas from pure research to applied and professional. The ASM was through state branches and can be easily progressed to a national formed more than half a century ago to promote the discipline of meeting if the idea demands it. The relationships between states microbiology and its role is now more important than ever. The and the utility of the Visiting Speaker Program has been enhanced national leadership is conscious of the need to adapt and change by a clearer pathway for VSP engagement, available on the website and has been moving in the past few years to do so, with gathering (http://www.theasm.org.au/events/visiting-speakers-program/) and momentum. One of our most important platforms is our national by more formal networking between the state branches, beginning meeting, developed to promote the exchange of ideas. The mem- in 2015. bership of the society is increasingly drawn to other conferences to A key part of our renaissance is a review of governance and meet their needs and this must be recognised and accommodated. organisational structure, which also begins in 2015, and the realign- Our approach to meetings including our national scientific meeting ment of Divisional Chair responsibilities. Nominations for new must evolve with it, as in comparable societies. Chairs for 2017 are actively sought and will be a key part of the repositioning of ASM. The revised Constitution is due to be released We now see increased centralisation and automation and much to all members shortly and to be voted on in the July 2015 AGM at greater incorporation of molecular diagnostics and other aspects of the Canberra meeting. biotechnology in industry, environmental and diagnostic microbiology. Accordingly, a national meeting under the auspices of the How can we participate in this rebirth and strengthen microbiology ASM to give a platform to discuss research and development in these as a discipline in a competitive environment? Read widely, talk freely fast-moving areas and to provide workshops for skill development with your colleagues inside and outside microbiology, and promote is being explored, initially focusing on clinical diagnostic microbi- the community by supporting ASM: join state branches and national ology. This follows the development of an additional ASM Travel council, apply for membership, including professional membership award for clinical microbiologists (http://www.theasm.org.au/ and Fellowship, develop new ideas for meetings with your branch or awards/asm-clinical-microbiology-travel-award/) and the Lyn Gilbert national office, and honour your colleagues by commending them Award (http://www.theasm.org.au/awards/asm-lyn-gilbert-award/) for awards and honorary memberships. for contributions in clinical microbiology, awarded for the first time The discipline of microbiology is somewhat different to what it was in 2014. when the Society began in 1959 and the Society must keep pace with Highly specialised meetings are important and essential for career it. ASM must better support progressive specialisation of the entire development and networking and for exchange of the latest infor- membership by supporting specialised meetings, some now long- mation between experts in fast-moving and competitive areas of established and completely autonomous and others that are yet be endeavour. Deep and narrow in scope by definition, attendance at conceived. We must at the same time enrich this with wider these to the exclusion of broader conversations may not meet the engagement and bring into our community those who do not see complete needs of early career microbiologists. We must therefore themselves as Microbiologists and yet work with us. The role of not only embrace and nurture new directions in microbiology but the ASM is not to push back against this natural evolution but to be open to ideas coming from outside that which we have long foster it.

2 10.1071/MA15001 MICROBIOLOGY AUSTRALIA * MARCH 2015 Guest Editorial

Mammalian microbiomes

discoveries in the late 1970s were paramount in facilitating mammalian microbiome research. The diversity of prokaryotes in a plethora of environments could be comprehended by applying massively parallel high throughput DNA sequencing methods (starting with 454 Life Sciences ‘pyrosequencing’ in 2005) to small subunit rRNA genes. Full genomes were determined in large numbers (currently of 31,241 prokaryotes), whole-genome phylogenies were reported, and the ‘meta-omics’ fields of endeavour were well and truly spawned into the general arena of the Linda L Blackall Microbiome. The giddy rate of progress in omics is difficult to keep Email: [email protected] pace with but critical questions about microbial function and dynamics (stable and mobile) and the chemical interplay between microbes and their hosts should be overriding drivers of their investigation, particu- Endothermic (an organism that maintains its body at a metabolically larly in mammals. Pondering the future of microbiome research, a 6 favourable temperature) amniotes (who lay their eggs on land or retain collection of authors recently reported on their individual opinions , the fertilised egg within the mother), also known as mammals, count and to quote from this paper: among their cohort the largest (whales) and the most intelligent (some Overall, future microbiome research regarding the mole- primates, cetaceans and elephants) animals on Earth. However, none of cules and mechanisms mediating interactions between the 5488 mammalian species live alone since they all support a complex members of microbial communities and their hosts should menagerie of microbes including prokaryotes (Bacteria and Archaea), lead to discovery of exciting new biology and transformative microbial eukaryotes, and viruses. That so-called ‘microbiome’ plays therapeutics. myriad roles ranging from the very well known and well studied The articles in this Microbiology Australia issue cover a broad range of (disease) through to provocative involvements (mood alteration and mammalian microbiome studies in Australia. The majority are on brain activity). Indeed even the microbiome has been subdivided by humans (oral and gut), but marine mammals (skin, gut and respiratory some into the bacteriome, the mycobiome and the virome. It is very tract including blowhole), ruminants and terrestrial Australian native timely that this issue be devoted to mammalian microbiomes since the animals (oral and gut) are also explored. The motivations for the host- study of microbiomes is going through an unprecedented revolution microbe studies in these papers cover the host species from both health due to current and projected capabilities to generate metagenome and well-being perspectives as well as from general ecological and sequences, determine metatranscriptomic, metaproteomic and meta- animal production improvement viewpoints. Monotreme microbial metabolomic information and crucially, analyse the deluge of data and ecology and potential biotechnological discoveries from the consump- interpret findings ecologically. The novel procedures are broadly in the tion of toxic diets (e.g. those high in essential oils like Eucalyptus spp.) economic realm of numerous researchers, but many do pose consid- should attract more attention and are of essential Australian relevance. erable technical challenges. The practical outcomes for host species and their environments are diverse. References Fundamental and applied host microbiome research began very early in 1. Hungate, R.E. (1966) The Rumen and its Microbes. Academic Press, New York. the history of microbiology. Indeed, Antonie van Leeuwenhoek 2. Hungate, R.E. and Macy, J. (1973) The roll-tube method for cultivation of strict – (1632–1723), ‘the father of microbiology’, observed and reported on anaerobes. Bulletins of the Ecological Research Committee 123 126. 3. Sanger, F. et al. (1977) DNA sequencing with chain-terminating inhibitors. Proc. what were large selenomonads from the human mouth in 1676. More Natl. Acad. Sci. USA 74, 5463–5467. doi:10.1073/pnas.74.12.5463 recently, Robert Hungate1 (1906–2004), ‘the father of rumen micro- 4. Sanger, F. et al. (1977) Nucleotide sequence of bacteriophage PHICHI174 DNA. ’ biology , developed critical techniques that allowed the study of anaer- Nature 265, 687–695. doi:10.1038/265687a0 – 2 obic microbes the roll tube technique . Using this method that he 5. Woese, C.R. and Fox, G.E. (1977) Phylogenetic structure of prokaryotic domain – meticulously described, he explored methanogenesis and cellulose primary kingdoms. Proc. Natl. Acad. Sci. USA 74, 5088–5090. doi:10.1073/ biochemistry (among other metabolic functions) in ruminants and the pnas.74.11.5088 microbial ecology of monkey and human guts. The practical outcomes 6. Waldor, M.K. et al. (2015) Where next for microbiome research? PLoS Biol. of improved milk, meat and wool production were major drivers to doi:10.1371/journal.pbio.1002050 rumen microbiology studies. Biography It has been nearly four decades since ‘Sanger DNA sequencing’ was Linda L Blackall is a microbial ecologist who has studied many introduced3, the first organism was sequenced4 and ribosomal RNA different complex microbial communities ranging from host associated analysis was used to determine the third domain of cellular life on Earth, through to free living in numerous environments. Her research has the Archaea5. Since the late 1970s, substantial method developments covered mammalian microbiomes spanning marsupials, humans, rumi- including polymerase chain reaction (PCR), improved acquisition of nants and horses and the methods used allow elucidation of massive DNA sequences (automated DNA sequencing) and their analyses have microbial complexity and function in these diverse biomes. She is a occurred. The first decade of this century was part of that method Professor of Biosciences at Swinburne University of Technology in the improvement and subsequent data eruption. These fundamental Faculty of Science, Engineering and Technology.

MICROBIOLOGY AUSTRALIA * MARCH 2015 10.1071/MA15002 3 In Focus

Methane matters in animals and man: from beginning to end

Emily Hoedt A, Paul Evans B, Stuart Denman C, Chris McSweeney C, Paraic ÓCuív D and Mark Morrison D,E ASchool of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Qld 4072, Australia BAutralian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Qld 4072, Australia CCSIRO Agriculture, Queensland Bioscience Precinct, St Lucia, Qld 4067, Australia DUniversity of Queensland Diamantina Institute, Translational Research Institute, Woolloongabba, Qld 4102, Australia ECorresponding author. Tel: +61 7 3443 6957, Fax: +61 7 3443 6966, Email: [email protected]

Methanogenic archaea resident in the mammalian gastroin- as well as the provisionally named ‘Methanoplasmatales’3.Members testinal tract have long been recognised for their capacity to of the methane club are very popular, invited to join virtually all participate in interspecies hydrogen transfer, with commen- anaerobic microbial communities and especially those where surate positive effects on plant biomass conversion. Howev- sulphate is limiting. Popular hangouts include moist soil biomes, fresh er, there is also still much to learn about these methanogenic water sediments and rice paddies, landfills, the gastrointestinal tracts archaea in regards to their metabolic versatility, host adap- of invertebrate and vertebrate animals, anaerobic lagoons and waste tation, and immunogenic properties that is of relevance to management facilities4–6. Indeed, the number and distribution of host health and nutrition. these hangouts have dramatically increased in recent decades in response to human population growth and urbanisation, as well as Methane, man and best laid plans the intensification of agriculture to feed a hungry world; but the The methane club has an exclusive membership, principally hangover has arrived. We are now being challenged to reduce methane restricted to the Domain Archaea and more specifically, the gas emissions, and in particular, methane emissions from livestock Euryarchaeota. Five orders of methanogens have long been recog- production systems, which are attributed with producing ~20% of nised: the Methanopyrales, Methanococcales, Methanobacteriales, global methane emissions7, in response to global concerns about our Methanomicrobiales, and Methanosarcinales1. However, the member- impacts on the environment and climate change. Additionally, the ship has recently been expanded to include the Methanocellales2 resurgent interest in the microbiota we share our body with, and their

4 10.1071/MA15003 MICROBIOLOGY AUSTRALIA * MARCH 2015 In Focus

impacts on our health and well-being, extends to the methane club8. methylotrophic methanogens in ‘low methane’ animals relates to For these reasons, there is a renewed interest in gut methanogens, their capacity for growth when the bacterial ruminotype favours less but is it more of the same or something new? We contend that there hydrogen production during fermentation12,17. is still much to be learned about members of the methane club and their behaviour in the digestive tracts of animals and man, from Differences downunder: the low methane beginning to end. emitting macropodids The macropodids (kangaroos, wallabies, pademelons and relatives) Separating the sheep from the goats: methane bear some similarity to ruminants in so far as their reliance on and livestock forestomach colonisation by microbes for plant biomass conversion and nutrient provision. In contrast, the foregut microbiota resident As herbivores, ruminants rely upon their microbial communities in these animals releases relatively low amounts of methane com- within the rumen-reticulum to not only deconstruct plant biomass, pared to sheep18,19. Although these observations were initially but provide the schemes of anaerobic fermentation necessary to proposed to reflect the absence of methanogenic archaea within support the formation of protein-yielding and energy-yielding the macropodid forestomach, several studies have now demon- nutrients such as microbial biomass and short chain fatty acids9,10. strated the presence of Methanobrevibacter, Methanosphaera, Methanogens have long been recognised to support these process- and ‘Methanoplasmatales’ archaea, albeit at numbers substantially es via minimising pH2, with the concept of ‘interspecies hydrogen less than found for ruminant livestock (~106 g.sample–1 c.f. ~108 g. transfer’ (IHT) first demonstrated by Bryant and Wolin11 using sample–1)6. Our group has now produced an axenic culture of culture based experiments with rumen bacteria and methanogens. Methanosphaera sp. (strain WGK6) from foregut digesta collected Much of the subsequent research focused on the taxonomic and from a Western grey kangaroo (Macropus fuliginosus). Like ecological variations among methanogen communities as affected the human strain Methanosphaera stadtmanae DSM-3091, WGK6 by diet, animal breed, and production system. In general terms, uses methanol for methane formation, energy production and these studies have shown that while autotrophic Methanobrevi- growth. However, the annotated draft sequence of the WGK6 bacter spp. are often numerically predominant there is also a genome suggests the macropodid isolate possesses some unique relatively diverse population of heterotrophic archaea present in features that may support a greater metabolic versatility than these animals6,12. In recent years, the application of ‘omics’ previously characterised from studies of the human strain. So approaches has provided new insights into the roles the archaea it seems that the adaptations to herbivory in the ‘low methane’ might play in rumen function. Poulsen et al. (2013) showed that emitting macropodids includes the maintenance of Methano- the reduced methane emissions from dairy cows fed rapeseed sphaera spp., which also seem to be present in greater abundance oil could be attributed to a selective suppression of the in ruminant animals individually confirmed to be ‘low methane’ ‘Methanoplasmatales’, with coincident decreases in transcripts emitters. encoding for methylotrophic methanogenesis from the rumen contents of these animals13. New Zealand and US-DOE researchers have also studied the rumen microbiota of sheep stratified with Humans and methanogens: a docile partnership respect to methane production, and demonstrated that the trait or secret frenemies? is heritable14. Using a combination of metagenomic and metatran- Methanogens are consistently identified from human subjects scriptomic methods they found no significant differences in total deemed healthy or suffering from disease; however the relation- methanogen numbers between the ‘low’ and ‘high’ methane ships between the diversity of methanogen community members producers, although there were differences in the relative abun- and the health status of the host are still unclear. Early studies dances of the methylotrophic Methanosphaera spp. (increased in determined that like other mammals the human large bowel ‘low methane’ sheep) and the hydrogenotrophic Methanobrevi- was colonised by hydrogenotrophic Methanobrevibacter spp. bacter gottschalkii clade (increased in ‘high methane’ sheep). The (principally Mbb. smithii) and the methylotrophic Methanosphaera metatranscriptomic data revealed that 7/10 genes coordinating spp. (principally Msp. stadtmanae20). More recently, the analysis of the hydrogenotrophic pathway were significantly increased in high human microbiota samples from subgingival, intestinal or vaginal methane producing sheep. Collectively, these findings suggest mucosae has further expanded the diversity of methanogenic that while the inhibition of select populations of methanogens can archaea to include a new species of Methanobrevibacter (Mbb. mitigate livestock methane emissions, it is also a heritable trait, oralis), as well as two isolates of methylotrophic archaea (Candi- suggesting host-mediated effects on the rumen microbiota. In that datus ‘Methanomethylophilus alvus’ and Methanomassiliicoccus context, ‘high methane’ emitting animals have been postulated luminyensis) affiliated with the newly defined order Methanoplas- to possess a longer retention of feed within the rumen as well as matales21,22. Interestingly, our own unpublished studies, as well alterations in the bacterial ‘ruminotype’ increasing the levels of as the findings of Poulsen et al.13, Dridi et al.21 and Borrel et al.22 ruminal hydrogen, with coordinate elevated expression of show these archaea are capable of using methylated amines arising genes encoding the hydrogenotrophic pathway and greater from phosphatidylcholine metabolism to support growth. In that methane yield15,16. It seems intuitive then to further suggest that context, establishment of the Methanoplasmatales in the human the increased relative abundance of hydrogen-dependent large bowel might be of clinical relevance for persons known to

MICROBIOLOGY AUSTRALIA * MARCH 2015 5 In Focus

possess relatively high levels of trimethylamine-oxide in blood, 10. Hobson, P.N. (1988) The Rumen Microbial Ecosystem, First edn. Elsevier Applied because of its association with cardiovascular disease pathogenesis Science, New York. fi (reviewed by Morrison23 and Brugère24). However, there is also 11. Bryant, M.P. and Wolin, M.J. (1975) Proceedings of the rst international congress of the international association of the microbiological society, Developmental mounting evidence from cross-sectional studies that variations in Microbiology E, Science Council of Japan, Tokyo, Japan. p. 297. archaeal communities at different body sites might impact human 12. Janssen, P.H. and Kirs, M. (2008) Structure of the archaeal community of the – health25 27. For instance, patients with periodontitis have been rumen. Appl. Environ. Microbiol. 74,3619–3625. doi:10.1128/AEM.02812-07 found to harbour large numbers of methanogenic archaea, in 13. Poulsen, M. et al. (2013) Methylotrophic methanogenic Thermoplasmata impli- addition to acetogenic and sulphate-reducing bacteria within sub- cated in reduced methane emissions from bovine rumen. Nat Commun 4, 1428. doi:10.1038/ncomms2432 gingival periodontal pockets28. Blais Lecours et al.29 also confirmed 14. Shi, W. et al. (2014) Methane yield phenotypes linked to differential gene that both Mbb. smithii and Msp. stadtmanae can be immunosti- expression in the sheep rumen microbiome. Genome Res. 24, 1517–1525. mulatory in animal models of respiratory disease, with the latter doi:10.1101/gr.168245.113 provoking a stronger immune response. Furthermore, Blais Lecours 15. Janssen, P.H. (2010) Influence of hydrogen on rumen methane formation and et al.30 reported that while the total numbers of methanogenic fermentation balances through microbial growth kinetics and fermentation – fl thermodynamics. Anim. Feed Sci. Technol. 160,1 22. doi:10.1016/j.anifeedsci. archaea are less in patients suffering from in ammatory bowel 2010.07.002 disease (IBD), the prevalence of Msp. stadtmanae was greater 16. Kittelmann, S. et al. (2014) Two different bacterial community types are linked in these patients, and healthy human subjects produced an with the low-methane emission trait in sheep. PLoS ONE 9, e103171. doi:10.1371/ antigen-specific IgG response to this archaeon. These results journal.pone.0103171 17. Attwood, G.T. . (2011) Exploring rumen methanogen genomes to identify suggest that Msp. stadtmanae prevalence and/or abundance may et al targets for methane mitigation strategies. Anim. Feed Sci. Technol. 166–167, be a biomarker of gut dysbiosis, being more prevalent in persons 65–75. doi:10.1016/j.anifeedsci.2011.04.004 with an altered ‘low hydrogen’ fermentation scheme. This hypoth- 18. Madsen, J. and Bertelsen, M.F. (2012) Methane production by red-necked walla- esis warrants more detailed examination as part of well-designed bies (Macropus rufogriseus). J. Anim. Sci. 90, 1364–1370. doi:10.2527/jas.2011- clinical studies of IBD and perhaps, other chronic inflammatory 4011 19. von Engelhardt, W. et al. (1978) Production of methane in two non-ruminant diseases. herbivores Comp. Biochem. Physiol. Part A. Physiol. 60,309–311. doi:10.1016/ 0300-9629(78)90254-2 Summary 20. Miller, T.L. and Wolin, M.J. (1985) Methanosphaera stadtmaniae gen. nov., sp. nov.: a species that forms methane by reducing methanol with hydrogen. Arch. Despite the widespread recognition of the roles methanogenic Microbiol. 141, 116–122. doi:10.1007/BF00423270 archaea play in gut environments, there is still much to learn about 21. Dridi, B. et al. (2012) Methanomassiliicoccus luminyensis gen. nov., sp. nov., a their metabolic versatility, host adaptation, and immunomodula- methanogenic archaeon isolated from human faeces. Int. J. Syst. Evol. Microbiol. tion. Recent research of the methylotrophic archaea from three 62, 1902–1907. doi:10.1099/ijs.0.033712-0 divergent mammalian hosts suggests that methane matters in 22. Borrel, G. et al. (2012) Genome sequence of ‘Candidatus Methanomethylophilus alvus’ Mx1201, a methanogenic archaeon from the human gut belonging to a animals and man, from beginning to end! seventh order of methanogens. J. Bacteriol. 194, 6944–6945. doi:10.1128/ JB.01867-12 References 23. Morrison, M. (2013) Looking large, to make more, out of gut metagenomics. Curr. Opin. Microbiol. 16, 630–635. doi:10.1016/j.mib.2013.10.003 1. Anderson, I. et al. (2009) Genomic characterization of methanomicrobiales reveals three classes of methanogens. PLoS ONE 4, e5797. doi:10.1371/journal. 24. Brugère, J.F. et al. (2014) Archaebiotics: proposed therapeutic use of archaea to – pone.0005797 prevent trimethylaminuria and cardiovascular disease. Gut Microbes 5,510. doi:10.4161/gmic.26749 2. Sakai, S. et al. (2008) Methanocella paludicola gen. nov., sp. nov., a methane- producing archaeon, the first isolate of the lineage ‘Rice Cluster I’, and proposal of 25. Furnari, M. et al. (2012) Reassessment of the role of methane production between the new archaeal order Methanocellales ord. nov. Int. J. Syst. Evol. Microbiol. 58, irritable bowel syndrome and functional constipation. J. Gastrointestin. Liver Dis. – 929–936. doi:10.1099/ijs.0.65571-0 21, 157 163. 3. Paul, K. et al. (2012) ‘Methanoplasmatales,’ Thermoplasmatales-related archaea 26. Pimentel, M. et al. (2003) Methane production during lactulose breath test is – in termite guts and other environments, are the seventh order of methanogens. associated with gastrointestinal disease presentation. Dig. Dis. Sci. 48,8692. Appl. Environ. Microbiol. 78,8245–8253. doi:10.1128/AEM.02193-12 doi:10.1023/A:1021738515885 4. Liu, Y. and Whitman, W.B. (2008) Metabolic, phylogenetic, and ecological diversity 27. Lepp, P.W. et al. (2004) Methanogenic Archaea and human periodontal disease. – of the methanogenic archaea. Ann. N. Y. Acad. Sci. 1125, 171–189. doi:10.1196/ Proc. Natl. Acad. Sci. USA 101, 6176 6181. doi:10.1073/pnas.0308766101 annals.1419.019 28. Vianna, M.E. et al. (2008) Quantitative analysis of three hydrogenotrophic micro- 5. Edwards, T. and McBride, B.C. (1975) New method for the isolation and identi- bial groups, methanogenic archaea, sulfate-reducing bacteria, and acetogenic fi fication of methanogenic bacteria. Appl. Microbiol. 29, 540–545. bacteria, within plaque bio lms associated with human periodontal disease. J. Bacteriol. 190, 3779–3785. doi:10.1128/JB.01861-07 6. Evans, P.N. et al. (2009) Community composition and density of methanogens in the foregut of the Tammar wallaby (Macropus eugenii). Appl. Environ. 29. Blais Lecours, P. et al. (2011) Immunogenic properties of archaeal species found Microbiol. 75, 2598–2602. doi:10.1128/AEM.02436-08 in bioaerosols. PLoS ONE 6, e23326. doi:10.1371/journal.pone.0023326 7. Lowe, D.C. (2006) Global change: a green source of surprise. Nature 439, 30. Blais Lecours, P. et al. (2014) Increased prevalence of Methanosphaera stadtma- fl 148–149. doi:10.1038/439148a nae in in ammatory bowel diseases. PLoS ONE 9, e87734. doi:10.1371/journal. pone.0087734 8. Samuel, B.S. et al. (2007) Genomic and metabolic adaptations of Methanobre- vibacter smithii to the human gut. Proc. Natl. Acad. Sci. USA 104, 10643–10648. doi:10.1073/pnas.0704189104 Biographies 9. Karasov, W.H. and Carey, H.V. (2009) Metabolic teamwork between gut microbes Emily Hoedt is a PhD student at The University of Queensland and hosts. Microbe 4, 323–328. School of Chemistryand Molecular Biosciences, and is supervised by

6 MICROBIOLOGY AUSTRALIA * MARCH 2015 In Focus

Mark Morrison, Phil Hugenholtz and Gene Tyson. Her PhD studies molecular basis of hydrogenotropy in gut microbial ecosystems focus on functional and comparative studies of heterotrophic with emphasis on the function of methanogenic archaea. The aim is methanogens from different gut environments, supported by an to eventually modify the ecosystem to reduce methane emissions Australian Postgraduate Award and a top-up scholarship from Meat from ruminant livestock. and Livestock Australia. Paul Evans is a Postdoctoral Fellow, based at the Australia Centre Dr Páraic ÓCuív is a gut microbiologist at The University of for Ecogenomics at University of Queensland, Brisbane and his Queensland Diamantina Institute where he has a long standing research interests are related to microbial ecology in anaerobic interest in host-microbe interactions as they relate to the aetiology environments. His research involves examinations of novel of chronic gut diseases. He is an expert in gut microbiology, microbes from a range of environments including ruminants, per- microbial genetics and functional metagenomics and he currently mafrost soils and coal bed methane aquifers. Paul is speciﬁcally leads the isolation and functional characterisation of microorgan- interested in archaea that populate these anaerobic environments isms from the human gut as part of the Australian Healthy Micro- and how they interact with other members of the microbial com- biome Project. munity to produce energy and make their living. Professor Mark Morrison is trained as a microbiologist with a Stuart Denman is a research scientist with the CSIRO in the speciﬁc interest in the role that microbes play in affecting the health Agriculture Flagship. He has been actively involved in projects that and well-being of humans and animals. After nearly 20 years within use molecular methods to detect and monitor key microbial popu- US academia, he returned to Australia in 2006 initially as a Science lations within the rumen. His current research focus is on microbial Leader within CSIRO, and now as Chair and Group Leader in metagenomics and uses advanced molecular and bioinformatic Microbial Biology and Metagenomics at The University of Queens- techniques to ascertain the interactions and functional changes land Diamantina Institute. His work since returning to Australia that take place in the rumen microbiome as they pertain to methane has emphasised the use of ‘omics’ technologies to produce new abatement strategies. insights into the microbial world, and from which, improved meth- Chris McSweeney is a Senior Principal Research Scientist at ods for monitoring and adjustment of the gut microbiota might CSIRO and leads research into the gut microbiology of livestock, be achieved; with the goal of improving host animal health and humans and native animals. His current research is focussed on the well-being.

MICROBIOLOGY AUSTRALIA * MARCH 2015 7 In Focus

The marine mammal microbiome: current knowledge and future directions

Tiffanie M NelsonA,F, Amy ApprillB, Janet MannC, Tracey L RogersDand Mark V BrownD,E ADepartment of Animal and Range Sciences, Montana State University, Bozeman, MT 59715, USA BWoods Hole Oceanographic Institution, 266 Woods Hole Road, Mailstop #4, Woods Hole, MA 02543, USA CGeorgetown University, Regents Hall 516, Washington, DC 20057, USA DEvolution and Ecology Research Centre, University of New South Wales, Kensington, NSW 2052, Australia ESchool of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2052, Australia FCorresponding author. Tel: +1 406 539 6898, Email: [email protected]

Marine mammals are globally significant because of their Recent investigations have highlighted the capacity of the micro- sensitivity to environmental change and threatened status, biome to act strongly and significantly in maintaining host health often serving as ‘ecosystem sentinels’1. Disease is a major with a vital role in disease manifestation and immune system cause of marine mammal population decline and the role of function2,3. Members of the microbial community can directly the microbiome in disease has generated considerable influence the progression of a disease via infection and also mod- interest. Recent research in humans has greatly enhanced ulate the host’s own immune system regulation and response4. our understanding of how the host-associated microbial Indeed the host’s microbial partners are essential to immune system community, the microbiome, affects host health. In this function. The microbiome has been observed to be species-specific review, we provide an overview of the extent of the marine in a variety of vertebrate hosts5–7 and is influenced by host phylog- mammal microbiome with a focus on whole community eny, as a result of millions of years of co-evolution8. Marine mammals characterisation using genomic methods. This research represent unique evolutionary lineages and investigations into their highlights the overlap in microbial communities between associated microbes will provide a deeper understanding of their geographically distinct species and populations of marine ecology and evolution. mammals, suggesting tight links between marine mammals and their microbial symbionts over millions of years of Marine mammals form a diverse group of 129 species in three evolution. An understanding of these links in both healthy orders, and of those, 28 are considered endangered or threatened9. and compromised hosts is essential to identifying at-risk Disease is one of the main causes of death in marine mammals and populations and making ecologically appropriate manage- some populations have suffered mass mortalities caused by bacterial ment decisions. We advocate further development of pathogens10. Bacteria exist as part of the normal, or even beneficial, innovative sampling and analytic techniques that advance flora associated with a host, fluctuating and changing with a host’s the field of microbial ecology of marine mammals. physiology and metabolism11. In mammals, disease can occur under

8 10.1071/MA15004 MICROBIOLOGY AUSTRALIA * MARCH 2015 In Focus 1 a number of different circumstances, most commonly on occasions 1 < < when the host’s immune system is compromised. For marine mammals, susceptibility to pathogens may be particularly elevated 1 1 < due to anthropogenic stressors such as depleted food resources, < habitat degradation and chemical or sound exposure12–15. Addi- Trichechus manatus latirostris tionally, succession events occurring after an initial bacterial infec- Sirenia 1 1 < < tion may lead to dysbiosis, and alterations in the host’s microbiome may be a better predictor of disease progression than following the

16 1 presence of individual pathogenic agents . Hence, we need to < dugong Dugong Manatee Dugong establish baseline data on microorganisms commonly associated with marine mammals in order to detect anomalies. In the last 10 < cinera decade genomic sequencing technologies have provided a previ- sea lion ously unrecognised diversity of microorganisms in numerous 1 diverse habitats. In this brief review we highlight the current <

knowledge of the microbial composition in associations with marine ibraries (CL); pyrosequencing (P); metagenomic sequencing (M). mammals with a focus on whole community characterisation. 1 14 8 < <

Skin microbiome Australian fur seal Australian Arctocephalus pusillus doriferus Neophoca 1 Skin, as the largest organ of mammals, serves as a thick physical < barrier that provides defense against the surrounding marine environment. Marine mammal skin is prone to lesions and disor- 1 < seal Grey grypus

ders, however the role of microorganisms in these conditions is Halichoerus still largely unresolved and knowledge is primarily founded on Carnivora

17 1 < cultivation-based studies . The recent application of cultivation- seal Phoca vitulina Harbour independent sequencing-survey approaches to humpback whale (Megaptera novaeangliae) skin has demonstrated that a unique seal cristata ecosystem of microbes resides on the skin surface (Table 1), which Hooded differs from the community present in seawater18. 69114441118117 59002 Among populations of humpback whales surveyed in diverse geo- Pup NR NR NR 9 m pup 6 m pup 2 m pup NR Adult Adult Sub-adult Calf seal

graphic regions, two genera of bacteria (Bacteroidetes genus Tena- Sothern elephant females born in captivity. cibaculum and Gammaproteobacteria genus Psychrobacter) were Mirounga leonina Cystophora sub-adult found to be cosmopolitan and abundant associates on humpback 26 whale skin . Scanning electron microscopy of humpback whale T. aduncus to 26 seal skin revealed a rich layer of microbial cells on the skin surface , but leptonyx as humpback whales regularly undergo skin sloughing through both 27 28 behavioural and physiological activities it is possible that the 11320621 < NR Adult Adult and T. truncatus robust Tenacibaculum and Psychrobacter cells may have some T. truncatus Hydrurga means to maintain their residence on the whale skin and could , A

provide beneﬁts to their host. Sequencing survey-based data also hybrid Sub-adult truncatus, T. aduncus demonstrate differences between the skin bacterial associates of 18,26 11 healthy and health-compromised humpbacks . Additional data 1 1 5 4443182250768387838083797971 < < on and study of the skin microbiome might potentially improve our ability to assess health status among free-ranging marine mammals, 1 19 19 19 20 7 7 7 21 21 21 22 22 22 23 24 25 25 25 < in particular cetaceans. Megaptera noveangliae Turisops

Gut microbiome Fusobacteria Firmicutes 1 The gastrointestinal tract is home to an abundant community of BacteroidetesProteobacteria 40 60 63 36 1 50 34 60 8 31 21 15 14 68 49 24 10 6 4 2 15 17 19 26 microorganisms. The gut microbiome plays a signiﬁcant role in food breakdown and digestion, the production of essential vitamins and Number of individualsMethodologyReference 51 P 6 P 24 P 4 CL 12 P 18 P P CL CL CL P P P M CL P P P Age groupSampleBacterial phyla (% of community) Adult Skin Calf Skin Adult and Blow Blow Faeces Faeces Faeces Colon Colon Colon Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Species Common name Humpback whale Bottlenose dolphin Leopard Sub-Order Mysticeti Odontoceti Phocidae Pinnipedia Order Cetacea

3 Hybrid bottlenose dolphin refers to individuals sired by minerals and regulation of the immune system . In young mammals, Table 1. Relative abundance of bacterial phyla compared between known studies of marine mammal species and anatomical sites in healthy individuals Data summarised for theA dominant bacterial phyla across species and anatomical sites. Table data are as follows: not recorded (NR); month (m); clone l

MICROBIOLOGY AUSTRALIA * MARCH 2015 9 In Focus

the gut microbiome is required for full development of the immune (a) system and maturation of the gut29,30. Studies of the complete gut microbiome of marine mammals include leopard seals (Hydrurga leptonyx), southern elephant seals (Mirounga leonine), grey seals (Halichoerus grypus), hooded seals (Cystophora cristata), harbor seals (Phoca vitulina), Australian fur seals (Arctocephalus pusillus doriferus), Australian sea lions (Neophoca cinerea), Florida mana- tees (Trichecus manatus latirostris) and dugongs (Dugong dugong). Across all these species the gut microbiome is composed largely of Firmicutes, Bacteroidetes and Proteobacteria (Table 1). Diet and age have been identiﬁed as factors that shape the composition of the gut microbiome7,25.

Amongst the seals, the gut microbiome of pinnipeds has a greater abundance of the phylum Firmicutes compared with phocids (b) (Table 1). A ‘core’ group of microorganisms including the genera Ilyobacter, Psychrilyobacter, Fusobacterium, Bacteroides, Subdo- lingranulum, Sporobacter, Sutterella, Weisella, Anaerococcus and Campylobacter have been observed within phocid seals7,21,22 whilst their herbivorous relatives, within the order Sirenia, shared members from the order Clostridiales, including the genera Clostridium and Ruminococcus24,25,31. The presence of shared bacterial operational taxonomic units (OTUs) in multiple hosts from different studies highlights the strong phylogenetic inﬂuence on microbial assembly. Figure 1. Exhaled ‘blow’ samples provide access to respiratory microbiome, host DNA, hormones and associated metabolites. Bottlenose dolphins can be trained to exhale on demand allowing Respiratory microbiome collections to be made routinely as shown here by Jillian Wisse from Respiratory illnesses such as pneumonia are a major cause of the National Aquarium in Baltimore, Maryland, USA in captive dolphins (a) and Dr Ewa Krzyszczyk, collecting samples from wild bottlenose 32 mortality in both wild and captive marine mammals . The cetacean dolphins that visit a beach in Shark Bay, WA, Australia (b). Photo credit upper respiratory tract terminates in a blowhole, positioned at the monkeymiadolphins.org. top of the head. This feature is a unique adaptation to life in the microbiome of humans, and whose growth is enhanced by the marine environment, and allows airways to be effectively sealed off presence of carbon dioxide36, which occurs in high abundances at from seawater. Upon surfacing, cetaceans forcefully exhale and in the termination of the respiratory tract. Representatives from each the process eject a substance termed blow (also called condensed of DAC 1, 2, and 3 have been present in every bottlenose dolphin respiratory vapor or exhaled breath condensate). This material has surveyed thus far, although the majority of sequences are associated 33 been shown to harbour potential pathogens in whales and has also with DAC 3, indicating this is likely a ubiquitous and critical com- been used to characterise the normal respiratory-associated micro- ponent of the dolphin respiratory system. Other ‘core’ taxa asso- biome residing in the upper respiratory tract of bottlenose dol- ciated with the dolphin respiratory microbial community appear 19,20 phins (see collection methods in Figure 1). Members of the to include the Arcobacter, Hydrogenimonaceae, Halotalea, Aqui- bacterial genera Plesiomonas, Aeromonas, Escherichia, Clostridi- marina, Helococcus, Mycetocola, Methylococcus and Marinimi- um and Pseudomonas, Burkholdaria, Mycobacterium, Haemo- crobium19. Temporal analysis of captive dolphins suggests phylis, Streptococcus and Staphylococcus (including multiple community composition in healthy animals is quite stable resistant Staphylococcus aureus) have been detected in both and that individual dolphins harbour consistently unique microbial 34 20,33,35 sick/dead and healthy, free-ranging cetaceans . communities19.

Blow samples from both free-ranging Tursiops truncatus and captive T. aduncus and T. truncatus were dominated by three Sampling techniques novel dolphin associated clades (termed DAC 1, 2 and 3) within Sampling of material for microbiological analysis from marine the Cardiobacteraceae lineage of the Gammaproteobacteria19,20. mammals is logistically challenging (reviewed by Hunt et al.37), The Cardiobacteraceae are facultative anaerobic, Gram-negative hence the majority of information on microbial disease comes from rod-shaped cells, members of which form part of the commensal captive or stranded animals that are not necessarily representative of

10 MICROBIOLOGY AUSTRALIA * MARCH 2015 In Focus

the greater wild population. However, current sampling methods mortality in wild marine mammals, it has been linked to viruses, (see examples in Figures 1 and 2) still provide considerable insight including morbillivirus, phocine distemper and influenza virus51–55. into the microbiome of marine mammals. Capture by sedation or Despite these links being made there is really very little known restraint has been employed on smaller species such as seals and regarding the ecological role of viruses in marine mammal hosts. dolphins7,38,39 and has recently been used for some larger whales40. Further investigations into the factors responsible for shaping the However, there are few opportunities to sample using these meth- marine mammal microbiome need to be made. Designing studies ods. It is increasingly common to use biopsy darts for collection of that control for host variation will allow us to make headway in our skin and blubber samples for genetic and, now, microbiological understanding of disease manifestation. Studies that focus on the studies18,41. Permissions for biopsy sampling can be challenging for functionality of the microbiome will reveal the interactions between some species of marine mammals, and repeated samplings are often host and the microbial community23,56. In human subjects, similar not possible for the same individuals. In order to increase existing target investigations have allowed for the development of novel data on the marine mammal microbiome, logistically feasible, non- metabolites to treat and prevent disease57. Unlike humans, howev- or minimally-invasive sampling protocols that are easily reproduc- er, to access adequate biological material, strides need to be taken to ible and provide biological material suitable for a range of studies develop innovative and non-invasive techniques for the collection of are necessary. For example, respiratory blow can be used to relevant samples from wild populations. examine host DNA42 and hormone levels43,44 as well as respiratory associated microorganisms19,33,37, while non-invasively collected fecal samples can be used to study host DNA45, prey items46 and Acknowledgements the gut microbiome22,23. We thank Dr Ewa Krzyszczyk and Jillian Wisse for allowing us to use their photographs. Future research It appears likely that there are deep branching clades of bacteria References that are uniquely associated with marine mammals and have been 1. Moore, S.E. (2008) Marine mammals as ecosystem sentinels. J. Mammal. 89, conserved throughout the evolution of their hosts. Many bacterial 534–540. doi:10.1644/07-MAMM-S-312R1.1 sequences obtained from marine mammal studies have close rela- 2. Hooper, L.V. et al. (2002) How host-microbial interactions shape the nutrient environment of the mammalian intestine. Annu. Rev. Nutr. 22,283–307. tives that originate from other marine mammal species. This has doi:10.1146/annurev.nutr.22.011602.092259 fi signi cant implications for the transmission of disease amongst 3. Bäckhed, F. et al. (2005) Host-bacterial mutualism in the human intestine. Science these hosts. As they are usually highly social animals, there are 307,1915–1920. doi:10.1126/science.1104816 numerous opportunities for the transfer of microorganisms 4. Maynard, C.L. et al. (2012) Reciprocal interactions of the intestinal microbiota and immune system. Nature 489,231–241. doi:10.1038/nature11551 between individuals47. Diseases in marine mammals have also been 48,49 5. Yildirim, S. et al. (2010) Characterization of the fecal microbiome from non-human shown to have their roots in other mammals, including dogs and wild primates reveals species specific microbial communities. PLoS ONE 5, humans50. In many cases where disease has caused significant e13963. doi:10.1371/journal.pone.0013963 6. McKenzie, V.J. et al. (2012) Co-habiting amphibian species harbor unique skin bacterial communities in wild populations. ISME J. 6,588–596. doi:10.1038/ ismej.2011.129 7. Nelson, T.M. et al. (2013) Diet and phylogeny shape the gut microbiota of Antarctic seals: a comparison of wild and captive animals. Environ. Microbiol. 15, 1132–1145. doi:10.1111/1462-2920.12022 8. Ley, R.E. et al. (2008) Evolution of mammals and their gut microbes. Science 320, 1647–1651. doi:10.1126/science.1155725 9. Pompa, S. et al. (2011) Global distribution and conservation of marine mammals. Proc. Natl. Acad. Sci. USA 108, 13600–13605. doi:10.1073/pnas.1101525108 10. Waltzek, T.B. et al. (2012) Marine mammal zoonoses: a review of disease manifestations. Zoonoses Public Health 59,521–535. doi:10.1111/j.1863-2378. 2012.01492.x 11. Pamer, E.G. (2007) Immune responses to commensal and environmental microbes. Nat. Immunol. 8, 1173–1178. doi:10.1038/ni1526 12. Mos, L. et al. (2006) Chemical and biological pollution contribute to the immunological profiles of free-ranging harbor seals. Environ. Toxicol. Chem. 25, 3110–3117. doi:10.1897/06-027R.1 13. Fair, P.A. et al. (2013) Associations between perfluoroalkyl compounds and Figure 2. Collection of samples from wild marine mammals is logistically immune and clinical chemistry parameters in highly exposed bottlenose dolphins challenging. The collection of quality biological material with minimal (Tursiops truncatus). Environ. Toxicol. Chem. 32,736–746. doi:10.1002/etc.2122 impact on the animal requires the development of innovative sampling methods. This photo shows petri dishes attached to a modified pole for 14. Kight, C.R. and Swaddle, J.P. (2011) How and why environmental noise impacts the collection of exhaled ‘blow’ samples from a southern humpback animals: an integrative, mechanisticreview. Ecol. Lett. 14, 1052–1061. doi:10.1111/ whale off the coast of northeast Australia. Photo credit Tracey Rogers. j.1461-0248.2011.01664.x

MICROBIOLOGY AUSTRALIA * MARCH 2015 11 In Focus

15. Kannan, K. et al. (2007) A comparative analysis of polybrominated diphenyl 37. Hunt, K.E. et al. (2013) Overcoming the challenges of studying conservation ethers and polychlorinated biphenyls in southern sea otters that died of infectious physiology in large whales: a review of available methods. Conservation Physi- diseases and noninfectious causes. Arch. Environ. Contam. Toxicol. 53, 293–302. ology 1, cot006. doi:10.1093/conphys/cot006 doi:10.1007/s00244-006-0251-8 38. Ortiz, R.M. and Worthy, G.A.J. (2000) Effects of capture on adrenal steroid 16. Klepac-Ceraj, V. et al. (2010) Relationship between cystic fibrosis respiratory and vasopressin concentrations in free-ranging bottlenose dolphins (Tursiops tract bacterial communities and age, genotype, antibiotics and Pseudomonas truncatus). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 125,317–324. aeruginosa. Environ. Microbiol. 12, 1293–1303. doi:10.1111/j.1462-2920.2010. doi:10.1016/S1095-6433(00)00158-6 02173.x 39. Fair, P.A. et al. (2014) Stress response of wild bottlenose dolphins (Tursiops 17. Mouton, M. and Botha, A. (2012) Cutaneous lesions in cetaceans: an indicator of truncatus) during capture-release health assessment studies. Gen. Comp. Endo- ecosystem status? in New Approaches to the Study of Marine Mammals, crinol. 206, 203–212. doi:10.1016/j.ygcen.2014.07.002 A. Romero and E.O. Keith, Editors. InTech. 40. St Aubin, D.J. et al. (2001) Hematology and plasma chemistry as indicators of 18. Apprill, A. et al. (2011) Humpback whales harbour a combination of specific and health and ecological status in beluga whales, Delphinapterus leucas. Arctic 54, variable skin bacteria. Environ. Microbiol. Rep. 3, 223–232. doi:10.1111/ 317–331. doi:10.14430/arctic791 j.1758-2229.2010.00213.x 41. Palsbøll, P.J. et al. (1997) Genetic tagging of humpback whales. Nature 388, 19. Lima, N. et al. (2012) Temporal stability and species specificity in bacteria 767–769. doi:10.1038/42005 associated with the bottlenose dolphins respiratory system. Environ. Microbiol. 42. Frère, C.H. et al. (2010) Thar she blows! A novel method for DNA collection from – Rep. 4,89 96. doi:10.1111/j.1758-2229.2011.00306.x cetacean blow. PLoS ONE 5, e12299. doi:10.1371/journal.pone.0012299 20. Johnson, W.R. et al. (2009) Novel diversity of bacterial communities associated 43. Hogg, C.J. et al. (2005) Determination of testosterone in saliva and blow of with bottlenose dolphin upper respiratory tracts. Environ. Microbiol. Rep. 1, bottlenose dolphins (Tursiops truncatus) using liquid chromatography-mass – 555 562. doi:10.1111/j.1758-2229.2009.00080.x spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 814, 21. Glad, T. et al. (2010) Ecological characterisation of the colonic microbiota in arctic 339–346. doi:10.1016/j.jchromb.2004.10.058 – and sub-arctic seals. Microb. Ecol. 60, 320 330. doi:10.1007/s00248-010-9690-x 44. Hunt, K.E. et al. (2014) Detection of steroid and thyroid hormones via 22. Smith, S.C. et al. (2013) Age-related differences revealed in Australian fur seal immunoassay of North Atlantic right whale (Eubalaena glacialis) respiratory Arctocephalus pusillus doriferus gut microbiota. FEMS Microbiol. Ecol. 86, vapor. Mar. Mamm. Sci. 30, 796–809. doi:10.1111/mms.12073 – 246 255. doi:10.1111/1574-6941.12157 45. Green, M.L. et al. (2007) Noninvasive methodology for the sampling and extrac- 23. Lavery, T.J. et al. (2012) High nutrient transport and cycling potential revealed tion of DNA from free-ranging Atlantic spotted dolphins (Stenella frontalis). in the microbial metagenome of Australian sea lion (Neophoca cinerea) faeces. Mol. Ecol. Notes 7, 1287–1292. doi:10.1111/j.1471-8286.2007.01858.x PLoS ONE 7, e36478. doi:10.1371/journal.pone.0036478 46. Deagle, B.E. et al. (2009) Analysis of Australian fur seal diet by pyrosequencing 24. Tsukinowa, E. et al. (2008) Fecal microbiota of a dugong (Dugong dugong)in prey DNA in faeces. Mol. Ecol. 18, 2022–2038. doi:10.1111/j.1365-294X.2009. captivity at Toba Aquarium. J. Gen. Appl. Microbiol. 54,25–38. doi:10.2323/ 04158.x jgam.54.25 47. Lombardo, M. (2008) Access to mutualistic endosymbiotic microbes: an under- 25. Merson, S.D. et al. (2014) Variation in the hindgut microbial communities of appreciated benefit of group living. Behav. Ecol. Sociobiol. 62,479–497. the Florida manatee, Trichechus manatus latirostris over winter in Crystal River, doi:10.1007/s00265-007-0428-9 – Florida. FEMS Microbiol. Ecol. 87,601 615. doi:10.1111/1574-6941.12248 48. Butina, T.V. et al. (2010) Canine distemper virus diversity in Lake Baikal seal 26. Apprill, A. et al. (2014) Humpback whale populations share a core skin bacterial (Phoca sibirica) population. Vet. Microbiol. 144,192–197. doi:10.1016/j.vetmic. community: towards a health index for marine mammals? PLoS ONE 9, e90785. 2009.12.027 doi:10.1371/journal.pone.0090785 49. Greig, D.J. et al. (2014) Surveillance for zoonotic and selected pathogens in 27. Clapham, P.J. et al. (1993) High-energy behaviors in humpback whales as a source harbor seals Phoca vitulina from central California. Dis. Aquat. Organ. 111(2), of sloughed skin for molecular analysis. Mar. Mamm. Sci. 9, 213–220. doi:10.1111/ 93–106. doi:10.3354/dao02762 j.1748-7692.1993.tb00448.x 50. Osterhaus, A.D. (2000) Influenza B virus in seals. Science 288, 1051–1053. 28. Durban, J.W. and Pitman, R.L. (2012) Antarctic killer whales make rapid, round-trip doi:10.1126/science.288.5468.1051 movements to subtropical waters: evidence for physiological maintenance 51. Thompson, P.M. and Miller, D. (1992) Phocine distemper virus outbreak in the – migrations? Biol. Lett. 8,274 277. doi:10.1098/rsbl.2011.0875 Moray Firth common seal population: an estimate of mortality. Sci. Total Environ. 29. Palmer, C. et al. (2007) Development of the human infant intestinal microbiota. 115,57–65. doi:10.1016/0048-9697(92)90032-N PLoS Biol. 5, e177. doi:10.1371/journal.pbio.0050177 52. Van Bressem, M.F. et al. (2014) Cetacean morbillivirus: current knowledge and 30. Vael, C. and Desager, K. (2009) The importance of the development of the future directions. Viruses 6, 5145–5181. – intestinal microbiota in infancy. Curr. Opin. Pediatr. 21, 794 800. doi:10.1097/ 53. Pollack, J.D. (2001) Caspian seal die-off is caused by canine distemper virus. MOP.0b013e328332351b Trends Microbiol. 9, 108. doi:10.1016/S0966-842X(01)01988-6 31. Eigeland, K. (2012) Bacterial community structure in the hindgut of wild and 54. Anthony, S.J. et al. (2012) Emergence of fatal avian influenza in New England – captive dugongs (Dugong dugon). Aquat. Mamm. 38, 402 411. doi:10.1578/ harbor seals. MBio 3, e00166–12. doi:10.1128/mBio.00166-12 AM.38.4.2012.402 55. Ramis, A.J. et al. (2012) Influenza A and B virus attachment to respiratory tract in 32. Venn-Watson, S. et al. (2012) Thirty year retrospective evaluation of pneumonia marine mammals. Emerg. Infect. Dis. 18, 817–820. doi:10.3201/eid1805.111828 in a bottlenose dolphin Tursiops truncatus population. Dis. Aquat. Organ. 99, 56. Stewart, J.R. et al. (2014) Survey of antibiotic-resistant bacteria isolated from 237–242. doi:10.3354/dao02471 bottlenose dolphins Tursiops truncatus in the southeastern USA. Dis. Aquat. 33. Acevedo-Whitehouse, K. et al. (2010) A novel non-invasive tool for disease Organ. 108,91–102. doi:10.3354/dao02705 surveillance of free-ranging whales and its relevance to conservation programs. 57. Donia, M.S. et al. (2014) A systematic analysis of biosynthetic gene clusters in the Anim. Conserv. 13,217–225. doi:10.1111/j.1469-1795.2009.00326.x human microbiome reveals a common family of antibiotics. Cell 158, 1402–1414. 34. Buck, C.D. and Schroeder, J.P. (1990) Public health significance of marine doi:10.1016/j.cell.2014.08.032 mammal disease,inHandbook of Marine Mammal Medicine, L.A. Dierauf, Editor. CRC Press, Boca Raton, FL. pp. 163–173. 35. Morris, P.J. et al. (2011) Isolation of culturable microorganisms from free-ranging Biographies bottlenose dolphins (Tursiops truncatus) from the southeastern United States. Tiffanie Nelson is a researcher from Australia currently undertak- Vet. Microbiol. 148,440–447. doi:10.1016/j.vetmic.2010.08.025 ing a postdoctoral fellowship at Montana State University, Bozeman, 36. Savage, D.D. et al. (1977) Cardiobacterium hominis endocarditis: description of two patients and characterization of the organism. J. Clin. Microbiol. 5,75–80. USA. Tiff is a microbial ecologist, who focuses on the microbiome

12 MICROBIOLOGY AUSTRALIA * MARCH 2015 In Focus

of marine mammals as well as humans and environmental dolphins throughout their lives and includes an international team samples. Her interests are in health and disease associated with on three continents where each group studies different aspects of the microbiome. Tiff’s current project is investigating the vaginal delphinid biology. tract microbiome of women in relation to bacterial vaginosis using both culture-dependant and -independent methods. Tracey Rogers is associate professor at the University of New South Wales. Tracey works across a diverse range of research fields Amy Apprill is a researcher at the Woods Hole Oceanographic with many years of experience working in Antarctica with marine Institution in Massachusetts, USA. Amy is a marine microbiologist mammals. The common theme in Tracey’s research is in attempting researching questions that focus on the contribution of microorgan- to understand how mammals respond to change. Tracey uses isms to the health and ecology of marine animals. Amy is also multidisciplinary approaches to understand the ecology of mam- interested in how animal-associated microbes reflect the alterations mals. Most of her work uses models and techniques with captive occurring in their surrounding marine environment. Her current populations for applications in field settings. Other techniques research uses a combination of field measurements and observa- include stable isotope analysis, satellite telemetry and acoustics. tions and laboratory experiments and relies on diverse methodology (cultivation, genomic, metagenomic and bioinformatic) to examine Mark Brown is a senior research fellow at the University of the microbiomes of reef-building corals and marine mammals. New South Wales, Sydney, Australia. He has extensive expertise in Janet Mann is professor of biology and psychology and vice research that focuses on microbes (Bacteria, Archaea and microbial provost for research at Georgetown University, Washington DC, Eukaryotes), primarily from marine environments. Mark’s main USA. Janet has expertise in the field of animal behavior with interest is in investigating how microbes interact with each other extensive research focusing on marine mammals. Her work has and their environment to form communities that sustain critical focused on social networks, female reproduction, calf development, ecosystem processes. His current research couples innovative life history, conservation, tool-use, social learning and culture in situ sampling methods, genetic tools, bioinformatics and eco- among bottlenose dolphins in Shark Bay, Australia. Her long-term logical theory to elucidate and predict the form, function and study ‘The Shark Bay Dolphin Research Project’, tracks over 1600 impact of microbes in rapidly changing ecosystems.

MICROBIOLOGY AUSTRALIA * MARCH 2015 13 In Focus

The role of the gut microbiome in host systems

Clarissa FebiniaA, Connie HaA, Chau Le A and Andrew Holmes A,B ASchool of Molecular Bioscience and Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia BCorresponding author. Email: [email protected]

The presence of microbes exerts such a profound influence The past decade has seen a dramatic, and ongoing, revision of this on animals that they are best considered holobionts – an view with recognition that those microbes that form communities organism comprised of multiple biological partners. The of stable composition at various body sites (our microbiomes) concept of dysbiosis is disease states that result from unde- influence many aspects of our postnatal development and physiol- sirable interactions between the partners in a holobiont. ogy. This is especially true of the gut microbiome. Many molecular mechanisms that link the gut microbiome The links between the gut microbiome and host physiological with host health and disease have now been established and properties are now known to be important to the pathophysiology these are giving rise to new insights in healthcare. In essence of metabolic and immunological diseases. Studies of germ-free (GF) these studies show that our microbiome is so closely inter- animals have demonstrated robust connections between the gut twined with our physiology that microbiome composition is microbiome and host development and physiology. These include reflective of many aspects of our health. Of special impor- roles in vascular development1 and immune cell maturation2,3. tance is recognition of the intersection between chronic diet A consequence of such developmental effects is that emergent habits and the microbiome in driving changes in our phys- aspects of animal health and physiology such as inflammatory tone4, iological state. In the foreseeable future it is likely micro- energy balance5,6, feeding behaviour and even mood and gross biome profiling will be a standard diagnostic test in diverse anatomy can differ in germ-free animals. Three key points that have areas of medicine and that interventions targeting the emerged from these studies are schematically represented as they microbiome will be developed. might apply to human biology in Figure 1. First, the existence of GF All animals are associated with microorganisms for the majority of animals indicates the presence of microbes is not essential for the their life, only embryonic stages are microbe-free. However the viable development and physiology of an animal. However, GF complexity of animal-microbe interactions and the nature of their animals are different, with significant constraints on their environ- outcomes vary. For some animals their associations with microbes mental fitness, including susceptibility to systemic infection should include obligate partnerships with a specific microbe that has they be exposed to pathogens and having additional nutritional obvious benefits for the animals life history (e.g. Coral:Zooxanthel- demands (Figure 1a). Second, if microbes are non-essential to lae, Squid:Vibrio, Aphid:Buchnera). For others, the animal may normal physiological processes of animals it is arguable that their have a specialised structure in which it receives obvious benefits most fundamental contribution to the animals state is alteration of from microbes (e.g. the rumen), but these arise via a community of how the animal system perceives and responds to its environment, many microbial species. In contrast microbes can also interact with both internal and external. Finally, both microbiome association animals to cause disease. For the majority of animals such specific studies and transplant studies show that different compositions of pathogens have historically been the focus of scientific attention. the microbiome are associated with different host states. Where the The remaining microbes were traditionally viewed as commensals. microbiome composition gives benefits to desirable host functions

14 10.1071/MA15005 MICROBIOLOGY AUSTRALIA * MARCH 2015 In Focus

(a) Monobiont (b) Holobiont

Figure 1. Schematic representation of current understanding of the impact of the presence of microbes on human health. The critical site for host-microbiome interaction is the intestinal interface where nutrients are absorbed and critical signals for regulation of homeostasis of the animal system originate. Animal studies have shown that microbes contribute directly to differences between monobionts (a) and holobionts (b) in the structure of the intestinal interface and in the breakdown of food. Differences in microbial composition can drive differences in animal health via immune and neuroendocrine signalling. such as improved nutrition on available foods or reduced immu- associated with health. This reflects a high degree of functional nopathology we may view the stable host-microbiome system as a redundancy in the gut community, whereby multiple microbes with healthy holobiont (Figure 1b1). Where the microbiome composi- similar functions comprise ‘guilds’ that have broadly similar eco- tion is stable, but results in undesirable host functions such as logical roles within the community. Thus either benefit or detriment impaired energy balance or inflammation we may view the holo- to the host system is typically an emergent property of the whole biont as being in a state of dysbiosis (Figure 1b2). microbial community. Details of the mechanisms by which variations in the gut community impact host nutrition and physiol- Although various studies have proposed some key beneficial ogy are now emerging. Broadly speaking microbes contribute to microbes for gut health (e.g. Faecalibacterium prausnitzii, Copro- nutrition through the production of metabolites and impact phys- coccus sp., Ruminococcus bromii, Bacteroidetes spp.), it is rare iology through both metabolites and structural components. that presence or absence of any one microbe is specifically Microbial conversion of digestion resistant carbohydrate to short

MICROBIOLOGY AUSTRALIA * MARCH 2015 15 In Focus

chain fatty acids (SCFAs) or production of vitamins both result in plans. In intervention the microbiome is itself the target of manip- increased capacity for the host system to extract nutrition from ulation (e.g. prebiotics or probiotics). Greater understanding of food6,7. The SCFAs also exert other effects in the host system, host-microbiome interactions can inform both routes through: particularly butyrate which is a primary energy source for colono- (1) identification of biomarkers of health or disease in microbiome cytes, and therefore important for maintaining epithelial health. association studies (e.g. cancer diagnostics17); (2) identification SCFAs also impact the function of other tissues and organs in of specific microbes or consortia of microbes that are capable of the host by acting as signalling molecules for G-coupled protein effecting change if introduced18–20; or (3) intervention in signalling receptors (e.g. GPR41, GPR43). Known regulatory roles of SCFAs pathways that derive from microbes21. Progress toward these include: appetite regulation, epigenetic state, gut motility, energy objectives could be achieved across a very wide range of diseases metabolism, endocrine functions, and immune regulation8-10. Since and conditions if microbial community profiling were broadly these SCFAs are primarily microbial metabolites it could be argued adopted as a standard test. However, standardised experimental that the host is monitoring the activity of its microbiome via protocols and metadata collection (e.g. sample collection, DNA metabolite sensors and integrating this information into homeo- extraction method22, diet formula) need to be implemented in static regulation. Similarly the host also monitors microbial presence order to discern patterns that are robust across geographically and via pattern recognition receptors and signalling pathways contribute culturally diverse populations23. to regulation of diverse aspects of immune and metabolic functions. fi Signi cantly, disruption of the key metabolite receptors (e.g. References GPR4111) or PRR receptors (e.g. TLR512) in mouse knockout models 1. Stappenbeck, T.S. et al. (2002) Developmental regulation of intestinal angiogen- is capable of eliciting disease states, highlighting the importance esis by indigenous microbes via Paneth cells. Proc. Natl. Acad. Sci. USA 99, of microbial signalling for dysbiosis. Collectively these observations 15451–15455. doi:10.1073/pnas.202604299 show that change in the nature or strength of various microbiome 2. Cebra, J.J. (1999) Influences of microbiota on intestinal immune system development. Am. J. Clin. Nutr. 69, 1046s–1051s. signals, rather than presence/absence of specific microbes, is the 3. Rhee, K.-J. et al. (2004) Role of commensal bacteria in development of gut- primary determinant of health or dysbiosis. Given this, in order associated lymphoid tissues and preimmune antibody repertoire. J. Immunol. to understand dysbioses we must ask what drives disturbance 172,1118–1124. doi:10.4049/jimmunol.172.2.1118 to this? 4. Lam, Y.Y. et al. (2011) Role of the gut in visceral fat inflammation and metabolic disorders. Obesity (Silver Spring) 19, 2113–2120. doi:10.1038/oby.2011.68 In wild type animals this signalling-based disturbance to host-micro- 5. Bäckhed, F. et al. (2004) The gut microbiota as an environmental factor that – biome interactions is thought to primarily arise through changes in regulates fat storage. Proc. Natl. Acad. Sci. USA 101, 15718 15723. doi:10.1073/ pnas.0407076101 microbial composition or activity. Since different microbes (e.g. 6. Turnbaugh, P.J. et al. (2006) An obesity-associated gut microbiome with Gram-positive vs Gram-negative) contain different microbe-associ- increased capacity for energy harvest. Nature 444, 1027–1031. doi:10.1038/ ated molecular patterns (MAMPs) they drive different PRR-signalling nature05414 pathways. Similarly since microbes differ in their capacities to 7. LeBlanc, J.G. et al. (2013) Bacteria as vitamin suppliers to their host: a gut microbiota perspective. Curr. Opin. Biotechnol. 24,160–168. doi:10.1016/ degrade macromolecules and which metabolites they produce, j.copbio.2012.08.005 changes in community composition will also drive changes in 8. Brown, A.J. et al. (2003) The orphan G protein-coupled receptors GPR41 and metabolite-signalling pathways. Although diverse factors including GPR43 are activated by propionate and other short chain carboxylic acids. J. Biol. Chem. 278, 11312–11319. doi:10.1074/jbc.M211609200 anatomical, genotypic, cultural and environmental factors can in- 9. Licciardi, P.V. et al. (2011) Histone deacetylase inhibition and dietary short-chain 13–15 fluence the gut microbial community , it is chronic diet patterns Fatty acids. ISRN Allergy 2011, 869647. doi:10.5402/2011/869647 that are thought to be the dominant factor, since what we eat, and 10. Kumar, H. et al. (2014) Gut microbiota as an epigenetic regulator: pilot study the pattern of food consumption, impact the availability of nutrients based on whole-genome methylation analysis. mBio 5. to gut bacteria for their growth and metabolism. Arguably, the key 11. Samuel, B.S. et al. (2008) Effects of the gut microbiota on host adiposity are modulated by the short-chain fatty-acid binding G protein-coupled receptor, insight is not the role of the microbes, but rather the role of diet as a Gpr41. Proc. Natl. Acad. Sci. USA 105, 16767–16772. doi:10.1073/pnas.08085 key modulator of the interaction between microbes and the 67105 host6,14,16. This reflects that major mechanisms of microbial influ- 12. Vijay-Kumar, M. et al. (2010) Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5. Science 328,228–231. doi:10.1126/ ence are via small molecules that are uniquely microbial cellular science.1179721 components or metabolites. In summary, the concepts of dysbiosis, 13. Ley, R.E. et al. (2008) Worlds within worlds: evolution of the vertebrate gut and animals as holobionts, are changing the way we view human microbiota. Nat. Rev. Microbiol. 6, 776–788. doi:10.1038/nrmicro1978 biology, especially modern diseases with a lifestyle component. 14. Swartz, T.D. et al. (2013) Preserved adiposity in the Fischer 344 rat devoid of gut microbiota. FASEB J. 27, 1701–1710. doi:10.1096/fj.12-221689 In general terms there are two routes to improve health via under- 15. Yatsunenko, T. et al. (2012) Human gut microbiome viewed across age and – standing of the microbiome; diagnostics and interventions. In geography. Nature 486, 222 227. 16. Fleissner, C.K. et al. (2010) Absence of intestinal microbiota does not protect diagnostics, microbiome signals are included in our evaluation of mice from diet-induced obesity. Br. J. Nutr. 104,919–929. doi:10.1017/ the host state to inform disease prognosis or intervention S0007114510001303

16 MICROBIOLOGY AUSTRALIA * MARCH 2015 In Focus

17. Zackular, J.P. et al. (2014) The human gut microbiome as a screening tool for for health. A particular focus is the relationship between our nutrient – colorectal cancer. Cancer Prev. Res. (Phila. Pa.) 7, 1112 1121. environment and its effect on host-microbiome interactions in 18. Lawley, T.D. . (2012) Targeted restoration of the intestinal microbiota with et al health. We gratefully acknowledge funding support from the ARC a simple, defined bacteriotherapy resolves relapsing Clostridium difficile disease in mice. PLoS Pathog. 8, e1002995. doi:10.1371/journal.ppat.1002995 and NHMRC. 19. Adamu, B.O. and Lawley, T.D. (2013) Bacteriotherapy for the treatment of intestinal dysbiosis caused by Clostridium difficile infection. Curr. Opin. Micro- Clarissa Febinia is a postgraduate student and the recipient of an biol. 16, 596–601. doi:10.1016/j.mib.2013.06.009 Australia Awards Scholarship with affiliations to the Eijkman Insti- 20. Atarashi, K. et al. (2013) Treg induction by a rationally selected mixture of tute for Molecular Biology, Jakarta. Her project is on the intersection Clostridia strains from the human microbiota. Nature 500, 232–236. doi:10.1038/nature12331 between cultural, genetic and diet factors in lifestyle disease. 21. Ryan, K.K. et al. (2014) FXR is a molecular target for the effects of vertical sleeve fi gastrectomy. Nature 509, 183–188. doi:10.1038/nature13135 Connie Ha is in the nal year of PhD studies on the mechanisms of 22. Kennedy, N.A. et al. (2014) The impact of different DNA extraction kits and diet-induced obesity and the recipient of an APRA. laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing. PLoS ONE 9, e88982. doi:10.1371/journal.pone.0088982 Chau Le is a PhD student working on the influence of gut microbes 23. Finucane, M.M. et al. (2014) A taxonomic signature of obesity in the microbiome? on regulation of feeding behaviour. She is the recipient of an APRA. Getting to the guts of the matter. PLoS ONE 9, e84689. doi:10.1371/journal. pone.0084689 Andrew Holmes is currently an Associate Professor in the School of Molecular Bioscience at the University of Sydney and leads Biographies research programs in the Charles Perkins Centre and Marie Bashir The authors are all members of the School of Molecular Bioscience Institute. He was the recipient of the 2006 Fenner Prize from the and the Microbiome node of the Charles Perkins Centre at the Australian Society for Microbiology. He has general interests in University of Sydney. Their research is focussed on understanding microbial diversity, its evolutionary origins and ecological applica- the dynamics of gut microbial community composition, the tions. He is a Senior Editor for Microbiology and The ISME Journal, mechanisms of host-microbe interaction in the gut and develop- and a member of the Editorial Boards of Applied and Environmen- ment of tools to enable management of the gut microbial ecosystem tal Microbiology and Environmental Microbiology.

MICROBIOLOGY AUSTRALIA * MARCH 2015 17 Under the Microscrope

Modulation of the rumen microbiome

Rosalind Gilbert A,B,D, Diane Ouwerkerk A,B,E and Athol Klieve B,C,F ARumen Ecology Unit, Department of Agriculture and Fisheries, Level 2A East, EcoSciences Precinct, Dutton Park, Qld 4102, Australia BQueensland Alliance for Agriculture and Food Innovation, University of Queensland, St Lucia, Qld 4067, Australia CSchool of Agriculture and Food Sciences, University of Queensland, Gatton Campus, Gatton, Qld 4343, Australia DCorresponding author. Tel: +61 7 3255 4289, Email: [email protected] ETel: + 61 7 3255 4291, Email: [email protected] FTel: +61 7 5460 1255, Email: [email protected]

A combination of animal genetics and the unique, enlarged plant material they may also utilise substrates produced by other fore-stomach of ruminants (rumen) enable domesticated microbes. The rumen microbial population includes bacteria that are ruminants to be sustained on forages and fibrous feedstuffs predominantly strict anaerobes with the capacity to be highly fibro- that would be otherwise indigestible. Ruminants can also lytic and proteolytic (generally of the phyla Bacteroidetes, Firmicutes utilise more easily digestible, high energy plant material and Proteobacteria), methanogenic archaea (phylum Euryarchaeota) such as grain, to achieve rapid increases in weight gain, and anaerobic fungi (fungal division Neocallimastigomycota)2,3. muscle bulk and in the case of dairy cows, high milk yields. These rumen microbes are predated on by populations of anaerobic Since the mid-1900s there has been a steady research effort protozoa (predominantly of the phylum Ciliophora)4,5 and viruses into understanding the digestive processes of ruminants, (predominantly dsDNA bacteriophages of the order Caudovirales)6. striving to maintain animal health and nutrition whilst maximising the productivity and environmental sustainabil- Rumen microbes friend or foe? Strategies ity of livestock production systems. This article describes for reducing plant toxicity, acidosis strategies developed to modulate the rumen microbial ecosystem, enabling the utilisation of plant feedstuffs that may and enteric methane otherwise be toxic and enhancing feed utilisation efficiency The modulation of rumen microbial populations has traditionally or controlling populations of specific rumen microbes, such focussed on strategies to improve feed digestibility and consequent- as those contributing to lactic acidosis and enteric methane ly increase overall animal productivity, reducing the time taken for emissions. It also traces advances in technologies that have ruminant livestock to reach market-weight specifications. Micro- enabled us to understand the underlying biological mechan- biologists and animal nutritionists have sought to determine the isms involved in the modulation of the rumen microbiome. impact of different diet formulations on rumen function and live- weight gains, investigating the effects of feedstuff pre-treatment The rumen microbial community employing either physical change (for example steam treatment, – The rumen contains a dense microbial community that actively rolling or flaking of grain7 9) or physical and chemical changes degrades plant material, providing the animal with energy via the through microbial and enzymatic pre-treatment (for example ensi- end-products of fermentation (short chain fatty acids) and protein in lage of fodder crops with or without the application of silage the form of microbial protein, which flows from the rumen into the inoculants10–12). Research has also sought to increase the environ- lower intestine1. Rumen microbes not only adhere to and degrade mental sustainability of livestock production by investigating the

18 10.1071/MA15006 MICROBIOLOGY AUSTRALIA * MARCH 2015 Under the Microscrope

viability of alternative feedstuffs such as those that may also be a by- impact on protozoal populations, it has proven difficult to complete- product of food production or industry (for example cotton seed ly remove protozoa from the rumen. meal13) or feeds that may be readily propagated on-farm (for Strategies have also been developed to control rumen populations example microalgae14,15). Strategies have also been developed to of the amylolytic bacterium Streptococcus bovis. Amylolytic, lactic allow cattle to utilise plant feedstuffs that may otherwise be toxic to acid-producing bacteria such as S. bovis, may over-proliferate in the the animal, for example the leguminous shrub Leucaena leucoce- rumen when cattle are fed high concentrate or high grain diets, phala may be propagated on-farm as a high-protein fodder crop contributing to the development of a condition known as lactic (Figure 1). Leucaena however, contains the toxic amino acid acidosis. S. bovis has been targeted through the application of mimosine and normal rumen microbial degradation results in the antibiotics such as monensin21 and phage therapies22. The strate- formation of the toxin 3-hydroxy-4(1H)-pyridone (DHP). The ac- gies developed to control S. bovis were largely undertaken between cumulation of these toxins leads to negative health implications the 1970s and 1990s and the relative importance of controlling these including hair loss, reduced live-weight gain and goitre. The solution organisms and their overall contribution to the development of to preventing the development of Leucaena toxicity arose from lactic acidosis has been cause for debate23,24. Feeding practices rumen microbes, as these toxins were first shown to be de-toxified avoiding sudden dietary changes to large quantities of fermentable by bacteria of the genera Synergistes isolated from the rumen of feral carbohydrates can prevent the development of acidosis25 and most goats16. A mixed microbial drench containing Synergistes jonesii is of the novel strategies to prevent rumen acidosis reported in the currently produced in Queensland forthe treatment of cattle grazing literature26,27 have not been either commercialised or implemented Leucaena17. within the livestock production industry.

In addition to strategies to improve feed breakdown in the rumen, In the past decade, investigations to modulate rumen microbial research has also been undertaken to specifically target and control populations have focused on strategies to reduce the amount of the certain rumen microbial populations including the rumen protozoa, potent greenhouse gas methane generated by livestock production. amylolytic bacteria, and more recently, the methanogenic archaea. Normal rumen microbial fermentation results in the accumulation Rumen protozoa may positively contribute to ruminant feed break- of hydrogen. This hydrogen may be utilised by acetogenic bacteria down18; however, as these eukaryotes actively graze on the rumen (for example of the genera Acetitomaculum, Eubacterium bacterial populations, their growth and proliferation within the and Blautia), however the majority of hydrogen is consumed by rumen may also contribute to the inefficient, intra-ruminal recycling populations of methanogenic archaea belonging to the genera of microbial protein5. Strategies to reduce rumen protozoa have Methanobrevibacter, Methanobacterium, Methanococcus, Metha- included the use of diet (high grain diets tend to reduce protozoal nomicrobium and Methanosaeta, with the methane produced lost populations), dietary additives such as the clay bentonite, nitrates to the animal via eructation28. Strategies currently in development and vaccination19,20. While these strategies have been shown to to control rumen methanogen populations include specific diets

Figure 1. Cattle herd grazing the leguminous shrub Leucaena leucocephala propagated on-farm in Northern Australia.

MICROBIOLOGY AUSTRALIA * MARCH 2015 19 Under the Microscrope

(high grain and/or oil), dietary additives (for example naturally- selected for further testing in order to increase starch utilisation occurring plant-derived compounds and synthetic anti-methano- (by the genera Ruminococcus, Prevotella and Butyrivibrio) and to genic compounds), animal breeding, phage-based therapies and assist with the prevention of lactic acid accumulation contributing – vaccination29 32. While several of these novel approaches have been to acidosis (M. elsdenii) in feedlot cattle. shown to be effective in reducing rumen methanogen populations, the majority are not yet at the stage of commercial application and Future directions adoption by the livestock production industry. The success of any strategies developed to modulate the microbial Feed supplements, probiotics and direct fed population of the rumen will always need to be underpinned by fundamental research efforts to understand how the strategies microbials impact on the baseline or ‘normal’ functioning of the rumen The Australian agricultural feed industry produces many feed sup- microbial ecosystem40. Addressing the gaps in current understand- plements designed and marketed to improve ruminant production ing of the rumen microbial ecosystem41 is therefore key to the efficiency, particularly for the dairy industry. Australian law requires development of new strategies to control the microbial populations. that all agricultural and veterinary chemical products sold in The rumen contains a very dense microbial ecosystem, end- Australia be registered by the Australian Pesticides and Veterinary products of microbial digestion, salts and plant material including Medicines Authority (APVMA, http://apvma.gov.au) and are listed on partially digested fibre, carbohydrate, and phenolic compounds the permits and PubCRIS database (https://portal.apvma.gov.au/ and as such, samples of rumen material often present unique pubcris). These feed supplements include direct fed microbials technical challenges. Rumen microbiologists have therefore always (DFM) or probiotics and may also incorporate additional enzymes been quick to adopt new developments in technology in order to (amylases, proteases), minerals and salts (selenium, potassium). more fully understand the complex microbial ecosystem of the There are approximately 30 formulations of probiotic microbes rumen. Early research efforts relied on microbial cultivation and available for use within Australia to enhance the overall digestive although the study of cultivated rumen microbes is important for efficiency of ruminants incorporating bacteria such as Bifidobacter- characterising microbial genera and elucidating their specific ium (including the species bifidum, longum and thermophilum), genetic and metabolic traits, culture-independent studies for the Lactobacillus (species acidophilus, delbrueckii subspecies bulgar- detection of specific microbes (real-time PCR assays) and commu- icus, plantarum and rhamnosus) and Enterococcus faecium. nity analysis based on the comparative phylogeny of the prokaryote In addition, a further 11 registered products are available that are 16S rRNA gene are often employed to ascertain the extent to which exclusively based on the yeast Saccharomyces cerevisiae. While the probiotic microbes survive and proliferate within the rumen31. survival and proliferation of these organisms in the rumen and the Rapid advances in high-throughput sequencing technologies have effect of these probiotic bacteria on rumen digestive processes also facilitated investigations into how probiotics can influence has been largely under-represented in the scientific literature, the both the community composition and functional gene capacity of probiotic effects of yeasts (S. cerevisiae) has been more extensively the rumen. Metagenomic studies of the rumen have progressed assessed33,34. Results can be highly variable between studies, how- current understanding of the functional gene potential of the rumen ever several investigations have established that the provision of microbial population enabling the in silico identification of enzymes these commercially available probiotics is most useful when applied involved in feed breakdown42. to young ruminants to accelerate the establishment of a healthy 35,36 gastrointestinal microflora . Probiotics may be used to exclude In the future, a greater reliance on gene sequence-based technol- undesirable zoonotic pathogens such as Escherichia coli O157 from ogies or ‘omics’ will lead to an increased understanding of the establishing in the ruminant gastrointestinal tract and may also interactions occurring between probiotics and the microbial popu- impact on the ruminant host immune system and feed breakdown lations indigenous to the ruminant gastrointestinal tract. This is of 36–38 efficiency . particular interest for the development and optimisation of new and more effective strategies for the modulation of the rumen While bacterial strains of rumen origin would be anticipated to microbiome. Development of new strategies, treatments and pro- survive and proliferate in the rumen and therefore have a selective biotics to enhance rumen feed utilisation efficiency, represents an advantage over microbes of non-rumen origin36, there are currently area of great potential for the Australian livestock industries and will no commercial formulations of rumen-derived probiotic bacteria enable the production of quality products to meet global demands. registered for use in Australia. The mixed microbial drench for Leucaena toxicity is the only APVMA approved rumen-derived References microbial treatment. There have however been several reports in 1. Bryant, M.P. (1959) Bacterial species in the rumen. Bacteriol. Rev. 23,125–153. the scientific literature of rumen-derived bacterial isolates being 2. Hungate, R.E. et al. (1964) The rumen bacteria and protozoa. Annu. Rev. examined for application as potential probiotics, for example, Microbiol. 18, 131–166. doi:10.1146/annurev.mi.18.100164.001023 fl Megasphaera eldenii, Ruminococcus sp., R. avefaciens, Prevo- 3. Orpin, C.G. (1975) Studies on the rumen flagellate Neocallimastix frontalis. 37,39 tella bryantii, Butyrivibrio fibrisolvens . These strains were J. Gen. Microbiol. 91, 249–262. doi:10.1099/00221287-91-2-249

20 MICROBIOLOGY AUSTRALIA * MARCH 2015 Under the Microscrope

4. Moon-van der Staay, S.Y. et al. (2014) The symbiotic intestinal ciliates and the 27. Styriak, I. et al. (1994) Isolation and characterisation of a new ruminal bacterio- evolution of their hosts. Eur. J. Protistol. 50, 166–173. doi:10.1016/j.ejop.2014. phage lytic to Streptococcus bovis. Curr. Microbiol. 28,355–358. doi:10.1007/ 01.004 BF01570201 5. Williams, A.G. (1986) Rumen holotrich ciliate protozoa. Microbiol. Rev. 50,25–49. 28. St-Pierre, B. and Wright, A.D.G. (2013) Diversity of gut methanogens in herbiv- – 6. Klieve, A.V. and Bauchop, T. (1988) Morphological diversity or ruminal bacter- orous animals. Animal 7,49 56. doi:10.1017/S1751731112000912 iophages from sheep and cattle. Appl. Environ. Microbiol. 54, 1637–1641. 29. Buddle, B.M. et al. (2011) Strategies to reduce methane emissions from – 7. Owens, F.N. et al. (1997) The effect of grain source and grain processing on farmed ruminants grazing on pasture. Vet. J. 188,11 17. doi:10.1016/j.tvjl.2010. performance of feedlot cattle: A review. J. Anim. Sci. 75,868–879. 02.019 8. Yang, W.Z. et al. (2001) Effects of grain processing, forage to concentrate ratio, and 30. Cottle, D.J. et al. (2011) Ruminant enteric methane mitigation: a review. Anim. – forage particle size on rumen pH and digestion by dairy cows. J. Dairy Sci. 84, Prod. Sci. 51, 491 514. doi:10.1071/AN10163 2203–2216. doi:10.3168/jds.S0022-0302(01)74667-X 31. Krause, D.O. et al. (2013) Board-invited review: rumen microbiology: leading – 9. Yang, W.Z. et al. (2013) Quality and precision processing of barley grain affected the way in microbial ecology. J. Anim. Sci. 91,331 341. doi:10.2527/jas.2012-5567 intake and digestibility of dry matter in feedlot steers. Can. J. Anim. Sci. 93, 32. Williams, Y.J. et al. (2009) A vaccine against rumen methanogens can alter the 251–260. doi:10.4141/cjas2012-132 composition of archaeal populations. Appl. Environ. Microbiol. 75, 1860–1866. 10. Beauchemin, K.A. et al. (2003) Effects of particle size of alfalfa-based dairy cow doi:10.1128/AEM.02453-08 diets on chewing activity, ruminal fermentation, and milk production. J. Dairy Sci. 33. Callaway, T. et al. (2012) Current status of practical applications: probiotics in dairy 86, 630–643. doi:10.3168/jds.S0022-0302(03)73641-8 cattle. In Direct-Fed Microbials and Prebiotics for Animals (Callaway, T.R. and – 11. Koenig, K. and Beauchemin, K. (2011) Optimum extent of barley grain processing Ricke, S.C., eds), pp. 121 135, Springer, New York. and barley silage proportion in feedlot cattle diets: growth, feed efficiency, and 34. Pinloche, E. et al. (2013) The effects of a probiotic yeast on the bacterial diversity fecal characteristics. Can. J. Anim. Sci. 91, 411–422. doi:10.4141/cjas2010-039 and population structure in the rumen of cattle. PLoS ONE 8, e67824. doi:10.1371/ 12. Zhao, Y.L. et al. (2015) Effects of volume weight, processing method and proces- journal.pone.0067824 sing index of barley grain on in situ digestibility of dry matter and starch in beef 35. AlZahal, O. et al. (2014) Use of a direct-fed microbial product as a supplement heifers. Anim. Feed Sci. Technol. 199,93–103. doi:10.1016/j.anifeedsci.2014. during the transition period in dairy cattle. J. Dairy Sci. 97, 7102–7114. 11.005 doi:10.3168/jds.2014-8248 13. Rogers, G.M. et al. (2002) Feeding cotton products to cattle. Vet. Clin. North Am. 36. Yeoman, C.J. and White, B.A. (2014) Gastrointestinal tract microbiota and pro- Food Anim. Pract. 18,267–294. doi:10.1016/S0749-0720(02)00020-8 biotics in production animals. Ann. Rev. Anim. Biosci. 2,469–486. doi:10.1146/ 14. Holman, B.W.B. and Malau-Aduli, A.E.O. (2013) Spirulina as a livestock supple- annurev-animal-022513-114149 ment and animal feed. J. Anim. Physiol. Anim. Nutr. (Berl.) 97, 615–623. 37. McAllister, T.A. et al. (2011) Review: the use of direct fed microbials to mitigate doi:10.1111/j.1439-0396.2012.01328.x pathogens and enhance production in cattle. Can. J. Anim. Sci. 91,193–211. 15. Panjaitan, T. et al. (2010) Effect of the concentration of Spirulina (Spirulina doi:10.4141/cjas10047 platensis) algae in the drinking water on water intake by cattle and the proportion 38. Newbold, C.J. and Hillman, K. (2004) Feed Supplements: enzymes, probiotics, of algae bypassing the rumen. Anim. Prod. Sci. 50, 405–409. doi:10.1071/AN09194 yeasts. In Encyclopedia of Animal Science (Pond, W.G., ed), pp. 376–378, CRC 16. Allison, M.J. et al. (1992) Synergistes jonesii, gen. nov., sp. nov.: a rumen bacterium Press. that degrades toxic pyridinediols. Syst. Appl. Microbiol. 15, 522–529. doi:10.1016/ 39. Klieve, A.V. et al. (2003) Establishing populations of Megasphaera elsdenii YE 34 S0723-2020(11)80111-6 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets. – 17. Klieve, A.V. et al. (2002) The production and storage of a fermentor-grown J. Appl. Microbiol. 95, 621 630. doi:10.1046/j.1365-2672.2003.02024.x bacterial culture containing Synergistes jonesii, for protecting cattle against 40. Russell, J.B. and Rychlik, J.L. (2001) Factors that alter rumen microbial ecology. mimosine and 3-hydroxy-4(1H)-pyridone toxicity from feeding on Leucaena Science 292, 1119–1122. doi:10.1126/science.1058830 – leucocephala. Aust. J. Agric. Res. 53,1 5. doi:10.1071/AR00121 41. Lima, F.S. et al. (2015) Prepartum and postpartum rumen fluid microbiomes: 18. Takenaka, A. et al. (2004) Fiber digestion by rumen ciliate protozoa. Microbes characterization and correlation with production traits in dairy cows. Appl. Environ. 19, 203–210. doi:10.1264/jsme2.19.203 Environ. Microbiol. 81, 1327–1337. doi:10.1128/AEM.03138-14 19. Hristov, A.N. et al. (2003) Evaluation of several potential bioactive agents for 42. Hess, M. et al. (2011) Metagenomic discovery of biomass-degrading genes reducing protozoal activity in vitro. Anim. Feed Sci. Technol. 105, 163–184. and genomes from cow rumen. Science 331, 463–467. doi:10.1126/science. doi:10.1016/S0377-8401(03)00060-9 1200387 20. Williams, Y.J. et al. (2014) Technical note: Protozoa-specific antibodies raised in sheep plasma bind to their target protozoa in the rumen. J. Anim. Sci. 92, Biographies 5757–5761. doi:10.2527/jas.2014-7873 21. Newbold, C.J. and Wallace, R.J. (1988) Effects of the ionophores monensin and Dr Rosalind Gilbert is a scientist with the Queensland tetronasin on simulated development of ruminal lactic acidosis in vitro. Appl. Department of Agriculture, Fisheries and Forestry, Rumen Ecology Environ. Microbiol. 54,2981–2985. Unit. Her research interests include rumen microbiology and the 22. Tarakanov, B.V. (1994) Regulation of microbial processes in the rumen by bacteriophages of Streptococcus bovis. Microbiol. 63, 373–378. (translated from role of phages in controlling rumen microbial populations. – Mikrobiologiya 363,657 667). Ms Diane Ouwerkerk is a Senior Molecular Biologist within the 23. Calsamiglia, S. et al. (2012) Is subacute ruminal acidosis a pH related problem? Rumen Ecology Unit of the Department of Agriculture, Fisheries Causes and tools for its control. Anim. Feed Sci. Technol. 172,42–50. doi:10.1016/ j.anifeedsci.2011.12.007 and Forestry based at the Ecosciences Precinct, Dutton Park, 24. Newbold, C.J. and Hillman, K. (2004) Feed Supplements: Enzymes, probiotics, Queensland. Her research interests include the use of molecular – yeasts. In Encyclopedia of Animal Science (Pond, W.G., ed), pp. 376 378, CRC techniques to investigate gut microbial ecosystems, particularly in Press. ruminants. 25. Krause, K.M. and Oetzel, G.R. (2006) Understanding and preventing subacute ruminal acidosis in dairy herds: a review. Anim. Feed Sci. Technol. 126, 215–236. Athol Klieve is the Associate Professor in Agricultural Microbiology doi:10.1016/j.anifeedsci.2005.08.004 at the University of Queensland. He has worked in rumen micro- 26. Klieve, A.V. et al. (2012) Persistence of orally administered Megasphaera elsdenii and Ruminococcus bromii in the rumen of beef cattle fed a high grain (barley) biology for 30 years and leads the collaborative UQ/DAFF Rumen diet. Anim. Prod. Sci. 52,297–304. doi:10.1071/AN11111 Ecology Unit.

MICROBIOLOGY AUSTRALIA * MARCH 2015 21 Under the Microscope

Polymicrobial nature of chronic oral disease

Stuart Dashper A, Helen Mitchell A, Geoff Adams A and Eric Reynolds A,B AOral Health Cooperative Research Centre, Melbourne Dental School, The University of Melbourne, Parkville, Vic. 3052, Australia BCorresponding author. Tel: +61 3 9341 1547, Fax: +61 3 9341 1596, Email: [email protected]

Recent microbiome studies have shown that the human oral half are officially named; however, draft genomes for approximately microbiome is composed of over 260 abundant bacterial half of these taxa are now available from the Human Oral Microbial species that predominantly live as polymicrobial biofilms Database (www.homd.org/)1. accreted to the non-shedding hard surfaces of the teeth. In 16S rRNA gene sequence surveys are providing a cost effective addition representatives of both Archaea and Fungi are means of studying microbiomes, identifying and enumerating a found in the oral cavity and there is considerable colonisa- relatively unbiased set of the prokaryotic species present, including tion of the soft tissues of the mouth. Most of these species are uncultivable species. This technique has been adopted for studying commensal and form complex biofilm communities that the oral microbiota; however, the results produced have not yet restrict the colonisation of the oral cavity by exogenous been definitive, with some studies finding huge variation across bacteria. Changes in the polymicrobial biofilm microenvi- individuals and limited or no differences between healthy and ronment such as those resulting from the effects of chronic diseased states. Many factors of the design and analysis of these inflammation for subgingival plaque, can lead to the emer- experiments can make it difficult to compare results between the gence of opportunistic pathogens resulting in dysbiosis and different studies, including pooling of samples, DNA extraction the development of chronic diseases such as periodontitis in method, marker gene or region used, primer sets, PCR conditions, a susceptible host. The application of microbiomic studies to sequencing platform, choice of taxonomic classifier and level of the analysis of these complex and dynamic communities in classification, clustering of read data into microbial groups, and the rigorously designed human clinical studies will provide statistical methods used for diversity analysis2. The composition of valuable mechanistic insight into the bacterial succession the healthy oral microbiota can certainly vary considerably across and complex interactions involved in the development of sites within the mouth, at the same site over time, and from person dysbiosis and disease. to person3,4. Although the 16S rRNA survey techniques are available The human oral cavity is the entry point of the gastrointestinal tract most clinical studies to date have been cross-sectional and have and offers a number of microenvironments that enable the prolif- investigated a limited number of bacterial species using either real eration of a wide range of largely commensal bacteria, the vast time PCR, checkerboard DNA-DNA hybridisation, or more recently, majority of which are endemic to the human oral cavity. Consider- the Human Oral Microbe Identification Microarray (HOMIM) able effort has been expended to identify the approximately 700 techniques. To compound these limitations samples taken from a prokaryote species that compose the total human oral microbiome. limited number of sites within the mouth are often pooled which Over one-third of these species remain uncultivated and less than can obscure comparative results and is not recommended for

22 10.1071/MA15007 MICROBIOLOGY AUSTRALIA * MARCH 2015 Under the Microscope

diverse communities such as the oral microbiome. Generating periodontitis at a site has been predicted by increases in the relative less information from a larger number of samples is much more proportions of P. gingivalis and/or T. denticola in subgingival informative than generating more information from a small number plaque at that site above threshold levels of 10–15%10. This and of samples, particularly for the classification of diseased and healthy more recent research demonstrating mutualism and synergistic states5. Furthermore, clinical sampling techniques may result in only virulence in animal models of periodontitis of multiple late colonis- part of the polymicrobial biofilm being collected; with the surface ing species found closely associated with disease progression in of the biofilm most closely associated with the host and disease humans has led to the more generally supported view of the process poorly represented in the sample. emergence of an opportunistic pathogenic polymicrobial biofilm as the trigger for disease progression in susceptible individuals fl Chronic periodontitis is an in ammatory disease of the supporting (Figure 1)11. A large number of disease-associated species have tissues of the teeth with an endogenous polymicrobial aetiology. been identified, consistent with the dysbiosis-hypothesis12, al- Over 47% of Americans over 30 (64.7 million adults) have chronic though further studies are required to differentiate commensal periodontitis, distributed as 8.7% mild, 30.0% moderate and 8.5% species that benefit from the disease process as opposed to those 6 severe . The prevalence and severity of periodontitis increases with that actually cause the disease. age, with more than 64% of adults aged 65 and over likely to have moderate or severe periodontitis. Numerous cross-sectional and In addition to the composition of the subgingival plaque polymi- fi longitudinal epidemiological studies have shown associations be- crobial bio lm, which will be revealed by microbiomic analyses, its tween periodontal diseases and a greater risk of certain systemic architecture is important as it has been shown that the late colonis- diseases and disorders, such as cardiovascular diseases, diabetes, ing opportunistic periodontal pathogens are found as microcolo- fi chronic kidney disease, metabolic syndrome, obesity, rheumatoid nies in the outer layer of the bio lm adjacent to the epithelium of an fl 13 fi arthritis, Alzheimer’s disease, pre-term and underweight births, and in amed periodontal pocket . These ndings indicate that the some cancers, particularly pancreatic, head and neck and oesopha- disproportionately large impact of the late colonising opportunistic geal cancers. These associations remain even after adjustment for pathogens in disease may be explained by their close proximity to fl medical and socio-economic confounding factors. the in amed tissue and resorbing alveolar bone.

Chronic periodontitis is episodic in nature with acute exacerbations The concepts of the roles of particular oral bacterial species in of destruction followed by periods of dormancy. Currently, diag- chronic periodontitis have changed over the past two decades but nosis of periodontitis is achieved retrospectively by clinical assess- there is wide consensus that anaerobic, proteolytic, amino acid ment of attachment loss. This loss of attachment is a result of fermenting species including Porphyromonas gingivalis, Trepone- pathogenic events that have already occurred at the diseased site, ma denticola and Tannerella forsythia play a crucial role in and any sampling at the time of diagnosis, may fail to identify those initiation and/or progression of disease. A process of bacterial species involved in active destruction. In addition different teeth succession in subgingival plaque has been described where mild within the same patient, as well as different sites around the same inflammation ofthe gingivaltissue (gingivitis) and the establishment of an appropriate microenvironment by early colonising bacteria 7,8 allows late colonisers to emerge as opportunistic pathogens . Chronic Periodontitis More recently P. gingivalis has been proposed to be a keystone pathogen that perturbs the ecological balance enabling the prolif- Progressive eration of other oral bacterial species resulting in the formation of a dysbiotic polymicrobial plaque, whilst remaining at very low levels Dysbiosis itself9. The keystone pathogen concept was adapted from conser- Pathogenic Biofilm Inflammation vation biology’s keystone species defining a low abundance species Homeostasis which has a disproportionately large effect on its environment. Commensal Biofilm Reduced Inflammation While this theory does help explain the major role of a few species in a complex polymicrobial biofilm, it is not entirely consistent Stable with prospective clinical trial data demonstrating that this bacterium, amongst others, proliferates during disease and can Oral Healthcrc represent a significant proportion of subgingival plaque bacteria at Figure 1. The changes in the subgingival plaque microbiome and the intimate association with the host inflammatory response that results in diseased sites. Furthermore the imminent progression of chronic a shift from a stable site to one that is undergoing disease progression.

MICROBIOLOGY AUSTRALIA * MARCH 2015 23 Under the Microscope

tooth can display varying degrees of disease severity, all undergoing Biographies periodontal disease progression at different rates. Therefore to Stuart Dashper is a Professor in the Oral Health Cooperative conclusively determine the polymicrobial aetiology of chronic peri- Research Centre and The Melbourne Dental School, The University odontitis and how opportunistic pathogens emerge and proliferate of Melbourne. Over the past 15 years he has developed a systems rigorously designed prospective human clinical trials coupled with biology approach to the study of chronic oral diseases that incor- microbiomic analyses are essential, followed by the testing of the porates the identification and characterisation of bacterial patho- bacterial species and communities identified in appropriate in vitro bionts, the composition and structures of the polymicrobial biofilm and animal models to determine their potential as polymicrobial communities in which they dwell, the molecular characterisation of biofilms to induce dysbiosis and disease. virulence-related traits and their interactions with other members of the bacterial community and the host. References 1. Dewhirst, F.E. et al. (2010) The human oral microbiome. J. Bacteriol. 192, 5002–5017. doi:10.1128/JB.00542-10 Helen Mitchell is a researcher with the Oral Health CRC and 2. Goodrich, J.K. et al. (2014) Conducting a microbiome study. Cell 158, 250–262. Masters of Science student in Bioinformatics at The University of doi:10.1016/j.cell.2014.06.037 Melbourne. She is undertaking comparative genomics of the peri- 3. Simón-Soro, Á. et al. (2013) Microbial geography of the oral cavity. J. Dent. Res. 92,616–621. doi:10.1177/0022034513488119 odontal pathobiont Porphyromonas gingivalis to determine viru- 4. Ge, X. et al. (2013) Oral microbiome of deep and shallow dental pockets in lence characteristics and assist in vaccine development. She is using chronic periodontitis. PLoS ONE 8, e65520. doi:10.1371/journal.pone.0065520 Next-Generation Sequencing techniques to determine bacterial 5. Hamady, M. and Knight, R. (2009) Microbial community profiling for human microbiome projects: tools, techniques, and challenges. Genome Res. 19, biomarkers of early childhood caries in saliva. 1141–1152. doi:10.1101/gr.085464.108 6. Eke, P.I. et al. (2012) Prevalence of periodontitis in adults in the United States: ’ 2009 and 2010. J. Dent. Res. 91, 914–920. doi:10.1177/0022034512457373 Geoff Adams has over 30 years experience as a biostatistician 7. Socransky, S.S. et al. (1998) Microbial complexes in subgingival plaque. J. Clin. and epidemiologist involved in consulting, teaching, and research. – Periodontol. 25, 134 144. doi:10.1111/j.1600-051X.1998.tb02419.x He has been employed by the Melbourne Dental School and the 8. Kolenbrander, P.E. et al. (2010) Oral multispecies biofilm development and the key role of cell–cell distance. Nat. Rev. Microbiol. 8, 471–480. doi:10.1038/ Oral Health CRC as a biostatistician and epidemiologist since 1999. nrmicro2381 Geoff manages the Oral and Systemic Disease program in the fi 9. Hajishengallis, G. et al. (2011) Low-abundance bio lm species orchestrates Oral Health CRC, which is investigating associations between peri- inflammatory periodontal disease through the commensal microbiota and com- plement. Cell Host Microbe 10, 497–506. doi:10.1016/j.chom.2011.10.006 odontal disease and various systemic conditions. 10. Byrne, S.J. et al. (2009) Progression of chronic periodontitis can be predicted by the levels of Porphyromonas gingivalis and Treponema denticola in subgingival plaque. Oral Microbiol. Immunol. 24, 469–477. doi:10.1111/j.1399-302X. Eric Reynolds AO PhD FICD FTSE FRACDS is a Melbourne 2009.00544.x Laureate Professor and CEO and Director of Research of the Oral 11. Tan, K.H. et al. (2014) Porphyromonas gingivalis and Treponema denticola Health CRC. He is also Head of the Oral Biology Section of the exhibit metabolic symbioses. PLoS Pathog. 10, e1003955. doi:10.1371/journal. ppat.1003955 Melbourne Dental School. He has been researching and teaching 12. Curtis, M.A. (2014) Periodontal microbiology—The lid’s off the box again. J. Dent. for over 30 years on the aetiology and prevention of the two major Res. 93,840–842. doi:10.1177/0022034514542469 oral diseases, dental caries and periodontal diseases, which are 13. Zijnge, V. et al. (2010) Oral biofilm architecture on natural teeth. PLoS ONE 5, e9321. doi:10.1371/journal.pone.0009321 associated with polymicrobial biofilms.

24 MICROBIOLOGY AUSTRALIA * MARCH 2015 Under the Microscope

Gastrointestinal microbiota, diet and brain functioning

Shakuntla Gondalia Centre for Human Andrew Scholey Psychopharmacology, School of Health Science, Swinburne Centre for Human University of Technology, Hawthorn, Psychopharmacology, School of Vic. 3122, Australia Health Science, Swinburne Tel: +61 3 9214 5100 University of Technology, Hawthorn, Email: [email protected] Vic. 3122, Australia

A growing interest for research in the relationship between different species3 from three major bacterial phyla, Bacteroidetes the gastrointestine (GI), GI microbiota, health and disease is (Gram-negative), Firmicutes (Gram-positive) and Actinobacteria due to the potential for research identifying intervention (Gram-positive). The proportion of these phyla in any individual strategies. Preclinical and clinical studies have indicated that depends upon that individual’s genetic makeup, dietary habits and initial colonisation of bacteria in the GI tract can affect the surrounding environment. individual’s health condition in later life. Diet is an influen- The initial inoculation then colonisation of the GI impacts the tial factor in modulating this complex ecosystem and con- GI microbiota throughout life and the dynamic microbial sequently can help to modulate physiological conditions. ecosystem is highly influenced by the surrounding environment The broader role of the GI microbiota in modulation of and dietary factors. Any modifications in this highly organised and pathology and physiology of various diseases has pointed complex ecosystem have the potential to influence the normal to the importance of bidirectional communication between physiological functions and are suspected to play a role in obesity, the brain and the GI microbiota in maintaining homeostasis. fatty liver disease, inflammation, diabetes and also psychological An association of diet with metabolic diseases is well known – conditions4 8. and there are dietary supplements reported to improve brain function and cognitive decline. In addition to the plausible Diet that impacts GI microbiota: from infancy mechanisms of inflammation and oxidative stress for psychological conditions, more research into the role of the GI to elderly microbiota in combination with dietary factors as a compo- Upon ageing, weakening of dentition, salivary function, digestion nent in psychological condition is warranted. From this and intestinal transit time may affect the intestinal microbiota9,10. work, targeted interventions could result. One manageable environmental factor is diet, which has been shown to significantly impact the GI microbial composition. The Although a link between GI function and health or disease in an types of bacteria present are dependent on the type of substrates individual has been determined, the underlying mechanisms are not available, with some bacteria prospering on a specific substrate clearly understood. The GI tract of a newborn is rapidly colonised by while others are unable to utilise that compound11. This selective microbiota during the birth process through maternal contact and substrate usage therefore enables modulation of the composition of from the surrounding environment. This microbial ecosystem sta- GI microbiota via the diet, which can occur at any stage of life. bilises in first 2–3 years of life and reaches maturity in the human However, diet in infancy can greatly affect the maturation process of adult1. By adulthood, the intestine contains approximately 1012 the microbial profile, gastrointestinal health and immune system. bacteria per gram of colonic content2, which is 10 times the number of cells in the human body. Based on 16S rRNA gene analyses, it was Human milk is normally the first dietary exposure to an infant. This estimated that an adult GI tract harbours between 500 to 1000 is fermented in the colon, stimulating the growth of specific bacteria

MICROBIOLOGY AUSTRALIA * MARCH 2015 10.1071/MA15008 25 Under the Microscope

(including Gram positive Bifidobacterium spp.). Infants fed with infection by preventing pathogen colonisation20, bioactivation of breast milk and those on milk formula developed and harboured beneficial constituents such as polyphenols21, detoxification of different and diverse bacterial populations in their GI tract12. More- xenobiotics and metabolism of luminal components leading to over, the composition of different milk formula facilitates different formation of a variety of metabolites such as short chain fatty acids types of bacterial colonisation. Infants fed with oligosaccharide (SCFA), vitamins and several gases11,22,23. enriched formula harbour higher counts of Bifidobacterium SCFAs such as n-butyrate, acetate and propionate act as key sources spp. and Lactobacillus spp. compared to infants fed on unsupple- of energy for tissues and promote cellular mechanisms that maintain mented formulae13. This prebiotic effect of infant food could be of a tissue integrity. When SCFAs reach the circulatory system, they major concern as it plays a role in shaping the GI microbiota that is impact immune function and inflammation8. SCFAs are also in- believed to influence an individual’s health for their entire life. A volved in host-microbe signalling and control of colonic pH with significant shift in the GI microbial ecology occurs when infants subsequent effects on microbial composition, intestinal motility and switch to a more solid and varied diet, including substantial reduc- epithelial cell proliferation24. Microbes have also been involved in tion in the percentage of Bifidobacterium spp. and Lactobacillus enzymatic degradation of complex substrates particularly many spp. in the total microbiota14. Throughout adulthood GI microbiota forms of polysaccharides from ingested food25. For example Bac- appears to become more stable15 as with more stable dietary habits. teroides thetaiotamicron, produces an array of enzymes for carbo- The final shift in composition and function of gut microbiota occurs hydrate breakdown26. During metabolism some gases such as during the older age of lifespan. In general, aging is associated with a methane, hydrogen, hydrogen sulfide and carbon dioxide are decline in physiological function including in the immune system produced within the GI tract. Excess production of these gases may and metabolism that consequently affects the microbial composi- cause GI problems such as bloating and pain. These gases may serve tion, or vice versa. Human age-related changes reported in the GI useful purposes however, it is debatable whether hydrogen sulfide microbial composition include a numerical decrease in Bifidobac- for example, is largely beneficial or harmful!27. teruim spp. and Lactobacillus spp. and an increase in Enterobac- teriaceae and obligate anaerobes such as Clostridium spp.16 Furthermore, GI microbiota can influence behaviour and brain function by influencing the expression of certain body chemicals Numerous dietary components have been identified as having such as hormons, neurotransmitters and neurotrophic factors. positive or negative effect on brain function and behaviour, this Commensal bacteria such as Bifidobacteria infantis can modulate effect can be improved by GI microbiota. Animal studies and human tryptophan metabolism, this suggest that GI microbiota may influ- clinical trials have well demonstrated the role of omega-3 polyun- ence the precursor pool for serotonin (5-HT)28. Animal studies have saturated fatty acid in normal brain functioning. Administration demonstrated that GI microbioa can also modulates brain chemistry of Bifidobacterium spp. in combination with a substrate for eico- such as Brain-Derived Neurotropic Factor (BDNF) expression in the sapentaenoic acid (EPA), alpha-linolenic acid results in increased hypothalamus and the brainstem29. BDNF is crucially involved in concentration of EPA in liver tissue and docosahexaenoic acid neurogenesis, brain development and neural circuit formation. in brain tissues17,18. In wild mice model, probiotics such as BDNF has also been recognised as an important antiobesity factor. L. helveticus prevented high fat diet-induced anxiety-like behav- GI microbiota maintain communication with the host using meta- iour19. Diet can modulate the GI microbial composition by provid- bolic, neural, immune and endocrine pathways. For health, homeo- ing a favourable environment while the contrary is also true that stasis between the GI microbiota and the host system is essential, GI microbiota can modulate the effect of diet and in turn host since any imbalance in this arrangement may result in a disease physiological and psychological function. condition4,5,7.

Activity of GI microbiota Interaction between GI microbiota and brain The GI microbiota can produce a vast range of metabolites and/or functioning structural components whose generation depends on the availabil- The evidence is increasing for a bidirectional route of communica- ity of nutrients and the luminal environment. These metabolites are tion between brain, gut and GI microbiota which use immune, subsequently taken up by GI tissues, potentially reach circulation neural and endocrine pathways and by this means inﬂuence gut- and other distant tissues and can be excreted in urine and breath. GI brain communication, brain function and even human behaviour4,5. microbiota have been linked to very important health functions such The top-down mechanism of the effect of stress and psychological as development and role of the immune system7, resistance to condition on the GI functions is known from extensive research, in

26 MICROBIOLOGY AUSTRALIA * MARCH 2015 Under the Microscope particular with Inflammatory Bowel Disease, Irritable Bowel Syn- References drome and Crohn’s disease. However, the role of GI microbiota in 1. Scholtens, P.A.M.J. et al. (2012) The early settlers: intestinal microbiology in early life. Annu. Rev. Food. Sci. Technol. 3,425–447. doi:10.1146/annurev-food-022811- brain functions such as stress, cognition and mood need to be 101120 explored more comprehensively. 2. Dominguez-Bello, M.G. et al. (2011) Development of the human gastrointestinal microbiota and insights from high-throughput sequencing. Gastroenterology The GI microbiota influences the release of the major neurotrans- 140,1713–1719. doi:10.1053/j.gastro.2011.02.011 mitters tryptophan, serotonin, endocannabinoid ligands, ghrelin 3. Hooper, L.V. and Gordon, J.I. (2001) Commensal host-bacterial relationships in the gut. Science 292, 1115–1118. doi:10.1126/science.1058709 and cholecystokinin, which can influence food intake, energy 4. Clemente, J.C. et al. (2012) The impact of the gut microbiota on human health: balance and some brain tasks such as emotion, cognition and motor an integrative view. Cell 148, 1258–1270. doi:10.1016/j.cell.2012.01.035 functions28,30. Changes in microbial composition and metabolism 5. Heijtz, R.D. et al. (2011) Normal gut microbiota modulates brain development – ‘ fl ’ and behavior. Proc. Natl. Acad. Sci. USA 108, 3047 3052. doi:10.1073/pnas. correlate with the concept of in amm-ageing , a low-grade chronic 1010529108 pro-inflammatory status as a common basis for a broad spectrum of 6. Desbonnet, L. et al. (2010) Effects of the probiotic Bifidobacterium infantis in age associated pathologies including cognitive decline and immu- the maternal separation model of depression. Neuroscience 170, 1179–1188. doi:10.1016/j.neuroscience.2010.08.005 nosenescence31. Age-associated changes may increase intestinal 7. Souza, D.G. et al. (2004) The essential role of the intestinal microbiota permeability and ease the passage of bacterial lipopolysaccharides in facilitating acute inflammatory responses. J. Immunol. 173, 4137–4146. doi:10.4049/jimmunol.173.6.4137 (LPS) into circulation, resulting in an elevated systemic LPS level. 8. Samuel, B.S. et al. (2008) Effects of the gut microbiota on host adiposity are When LPS binds to pattern recognition receptors such as toll-like modulated by the short-chain fatty-acid binding G protein-coupled receptor, receptor 4 on immune cells, there is induction of inflammation by Gpr41. Proc. Natl. Acad. Sci. USA 105,16767–16 772. doi:10.1073/pnas.08085 67105 production and release of cytokines, leukotrienes and prostaglan- 9. Lovat, L.B. (1996) Age related changes in gut physiology and nutritional status. 32 din . Animal studies with probiotic supplementation demonstrated Gut 38, 306–309. doi:10.1136/gut.38.3.306 that probiotics can normalise immune responses, reverse beha- 10. Claesson, M.J. et al. (2012) Gut microbiota composition correlates with diet and health in the elderly. Nature 488,178–184. doi:10.1038/nature11319 vioural deficits and restore basal noradrenaline level in response to 11. Cummings, J.H. and Macfarlane, G.T. (1991) The control and consequences of 6 stress . The probiotics also normalise central nervous system (CNS) bacterial fermentation in the human colon. J. Appl. Bacteriol. 70,443–459. biochemistry and improve behaviour in a mouse model of colitis, doi:10.1111/j.1365-2672.1991.tb02739.x 12. Harmsen, H.J.M. et al. (2000) Analysis of intestinal flora development in breast-fed through vagal nerve pathways for gut-brain communication33. Psy- and formula-fed infants by using molecular identification and detection methods. chological responses to the GI microbiome composition may be an J. Pediatr. Gastroenterol. Nutr. 30,61–67. doi:10.1097/00005176-200001000- 00019 important factor in understanding the increasing prevalence of 13. Penders, J. et al. (2006) Factors influencing the composition of the psychological conditions in the community and research into this intestinal microbiota in early infancy. Pediatrics 118,511–521. doi:10.1542/ topic should be promoted. peds.2005-2824 14. Sghir, A. et al. (2000) Quantification of bacterial groups within human fecal flora by oligonucleotide probe hybridization. Appl. Environ. Microbiol. 66, 2263–2266. Conclusion doi:10.1128/AEM.66.5.2263-2266.2000 Clinically, a psychological condition does not stand alone since there 15. Agans, R. et al. (2011) Distal gut microbiota of adolescent children is different from that of adults. FEMS Microbiol. Ecol. 77,404–412. doi:10.1111/j.1574- are frequently immune system and GI comorbidities. Antidepres- 6941.2011.01120.x sants are the commonest treatment, and as such are focused on a 16. Lakshminarayanan, B. et al. (2013) Prevalence and characterization of Clostridium top-down approach. However, growing evidence from increasing perfringens from the faecal microbiota of elderly irish subjects. J. Med. Microbiol. 62, 457–466. doi:10.1099/jmm.0.052258-0 numbers of animal studies and human trials suggest that the GI 17. Wall, R. et al. (2012) Contrasting effects of Bifidobacterium breve NCIMB 702258 microbiota composition can be correlated with the incidence of and Bifidobacterium breve DPC 6330 on the composition of murine brain fatty acids and gut microbiota. Am. J. Clin. Nutr. 95,1278–1287. doi:10.3945/ajcn. complex conditions such as cognitive decline, anxiety and depres- 111.026435 sion. Detailed clinical interpretation is warranted so that novel 18. Wall, R. et al. (2010) Impact of administered Bifidobacterium on murine host fatty – interventions in neuropsychological conditions can be employed. acid composition. Lipids 45, 429 436. doi:10.1007/s11745-010-3410-7 19. Ohland, C.L. et al. (2013) Effects of Lactobacillus helveticus on murine behavior Preclinical research combining detailed exploration of the GI micro- are dependent on diet and genotype and correlate with alterations in the biota in well-designed human cohort studies investigating the gut microbiome. Psychoneuroendocrinology 38, 1738–1747. doi:10.1016/ j.psyneuen.2013.02.008 impact of antibiotics, probiotics and diet on the brain and CNS 20. Wells, C.L. et al. (1988) Role of intestinal anaerobic bacteria in colonization functions will inform us of the importance of bottom-up mechan- resistance. Eur. J. Clin. Microbiol. Infect. Dis. 7,107–113. doi:10.1007/ isms in neuropsychological conditions. Further research in this BF01962194 emerging area will provide novel targets for interventions in psy- 21. Miene, C. et al. (2011) Impact of polyphenol metabolites produced by colonic microbiota on expression of COX-2 and GSTT2 in human colon cells (LT97). Nutr. chological disorders. Cancer 63, 653–662. doi:10.1080/01635581.2011.552157

MICROBIOLOGY AUSTRALIA * MARCH 2015 27 Under the Microscope

22. Donohoe, D.R. et al. (2011) The microbiome and butyrate regulate energy 33. Bercik, P. et al. (2011) The anxiolytic effect of Biﬁdobacterium longum ncc3001 metabolism and autophagy in the mammalian colon. Cell Metab. 13, 517–526. involves vagal pathways for gut–brain communication. Neurogastroenterol. Motil. doi:10.1016/j.cmet.2011.02.018 23, 1132–1139. doi:10.1111/j.1365-2982.2011.01796.x 23. Topping, D.L. and Clifton, P.M. (2001) Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Physiol. Rev. 81, Biographies 1031–1064. Shakuntla Gondalia is as Postdoctoral Fellow at Swinburne Uni- 24. Musso, G. et al. (2011) Interactions between gut microbiota and host metabolism predisposing to obesity and diabetes. Annu. Rev. Med. 62, 361–380. doi:10.1146/ versity of Technology, Victoria and her research interest incorpo- annurev-med-012510-175505 rates gastrointestinal microbial ecology and nutritional intervention 25. Cantarel, B.L. et al. (2012) Complex carbohydrate utilization by the healthy human microbiome. PLoS ONE 7, e28742. doi:10.1371/journal.pone.0028742 with the potential to improve the health and cognitive performance. 26. Xu, J. et al. (2003) A genomic view of the human-Bacteroides thetaiotaomicron Her research aims to better understand the effective mechanisms of symbiosis. Science 299, 2074–2076. doi:10.1126/science.1080029 GI microbiota, nutritional and dietary intervention on individual’s 27. Carbonero, F. et al. (2012) Microbial pathways in colonic sulfur metabolism physiological and psychological health condition. and links with health and disease. Front. Physiol. 3, 448. doi:10.3389/fphys. 2012.00448 Professor Andrew Scholey is director of the Centre for Human ﬁ 28. Desbonnet, L. et al. (2008) The probiotic Bi dobacteria infantis: an assessment Psychopharmacology at Swinburne University, Melbourne. He is a of potential antidepressant properties in the rat. J. Psychiatr. Res. 43, 164–174. doi:10.1016/j.jpsychires.2008.03.009 leading international researcher into the neurocognitive effects of 29. Schéle, E. et al. (2013) The gut microbiota reduces leptin sensitivity and the natural products, supplements and food components, having pub- expression of the obesity-suppressing neuropeptides proglucagon (GCG) and brain-derived neurotrophic factor (BDNF) in the central nervous system. lished over 160 peer-reviewed journal articles, books and book Endocrinology 154, 3643–3651. doi:10.1210/en.2012-2151 chapters. Scholey has been lead investigator in a series of studies 30. Gruninger, T.R. et al. (2007) Molecular signaling involved in regulating feeding into the human biobehavioural effects of natural products, and their and other mitivated behaviors. Mol. Neurobiol. 35,1–19. doi:10.1007/BF0270 0621 neurocognition-enhancing and anti-stress/anxiolytic properties

31. Franceschi, C. et al. (2007) Inflammaging and anti-inflammaging: a systemic (including first-into human neurocognitive assessment of Ginseng, perspective on aging and longevity emerged from studies in humans. Mech. Sage and Lemon balm among others). His current research focuses Ageing Dev. 128,92–105. doi:10.1016/j.mad.2006.11.016 on neuroimaging and biomarker techniques to better understand 32. Piya, M.K. et al. (2013) Metabolic endotoxaemia: is it more than just a gut feeling? Curr. Opin. Lipidol. 24,78–85. doi:10.1097/MOL.0b013e32835b4431 the mechanisms of cognitive enhancement.

28 MICROBIOLOGY AUSTRALIA * MARCH 2015 Under the Microscope

Marsupial oral cavity microbiome

Philip S Bird A,D, Wayne SJ Boardman B, Darren J Trott B and Linda L Blackall C AThe University of Queensland, School of Veterinary Science, Faculty of Science, Gatton, Qld 4343, Australia BThe University of Adelaide, School of Animal and Veterinary Sciences, Roseworthy, SA 5371, Australia CSwinburne University of Technology, School of Science, Faculty of Science, Engineering and Technology, Hawthorn, Vic. 3122, Australia DCorresponding author. Tel: +61 7 5460 1834, Fax: +61 7 5460 1922, Email: [email protected]

The oral microbiome of humans and animals will cause oral and reported in a letter to the Royal Society on 17 September 1683. disease within their lifetimes and include a large number of These first recorded observations of living bacteria showed ‘an endogenous cariogenic, periodontal and other opportunis- unbelievable great company of living animalcules and of enormous tic pathogens. Studies over many decades have attempted number of a variety of shapes and sizes’. Since then many oral to determine which bacteria are involved in oral diseases. prokaryotic species have been described. The Human Oral Micro- Earlier studies used exclusively culture-based methods. Now biome Database (www.homd.org/) has to-date >200 bacterial spe- culture-independent methods are being used to determine cies described from culture-dependent methods and approximately the composition of the microbiome in health and disease. 1,000 phylotypes detected by 16S rRNA gene sequencing of oral There have been limited numbers of studies of the marsupial samples from the human oral cavity using culture-independent microbiome and this report covers some of the research of methods9. A search of the International Journal of Systematic and those studies. Evolutionary Microbiology over the past 10 years for ‘oral’ revealed a host of novel bacterial species isolated from the oral cavity of Dental plaque, a natural oral biofilm is composed of many diverse humans and animals, including domesticated and wild (free-ranging bacterial species, some of which are involved in the aetiology of and captive) animals. In marsupials, novel microbial species were periodontal diseases (gingivitis and periodontitis)1. Factors such as revealed in the oral cavity of macropods associated with gingivitis indigenous bacteria, host immune system, diet, host susceptibility and oral necrobacillosis10. and time, interplay in these diseases2. There have been many studies determining which of the causative agent(s) initiate oral diseases in In domesticated and wild animals, using culture-dependent meth- humans and domesticated animals. Marsupials also have oral dis- ods, associations with disease and specific bacterial species have eases3,4 and culture-dependent studies have shown a range of been reported5. More recently, researchers have used high- bacteria can be isolated from the marsupial oral cavity5. However throughput DNA sequencing to study the oral microbiota of culture-based studies, while useful to enable precise characterisa- healthy cats8 and dogs7. In adult horses 67% of 203 operational tion of putative periodontopathogens, generally underestimate taxonomic units (OTUs) were recovered, with the most frequent microbial community diversity. Culture-independent methods, genera being Prevotella and Porphyromonas (T Chinkangsadarn, such as high-throughput DNA sequencing reveal a rich and diverse GJ Wilson, SW Corley and PS Bird, 2014, unpublished). In each case, bacterial community in the oral cavities of humans and companion the results revealed a rich and diverse bacterial community in animals6-8, and the marsupial microbiome should also be more much higher numbers than identified using culture- and cloning- thoroughly studied too. based studies. Therefore, in studies of animals with known oral The first bacteria ever to be seen under a microscope were the health status, health and disease could be correlated with the oral plaque bacteria taken from Antonie van Leeuwenhoek’s own teeth microbiota detected using these new technologies.

MICROBIOLOGY AUSTRALIA * MARCH 2015 10.1071/MA15009 29 Under the Microscope

We have shown that oral diseases such as gingivitis and periodontitis most abundant organism in mixed cultures. It also was isolated in can be found in a range of native Australian animals including high abundance from gingival margin samples taken from sites macropods, koalas (Phascolarctos cinereus), brushtail possums remote from the lumpy jaw lesions in 61% of the animals with (Trichosurus vulpecular) and bandicoots (Isoodon macrourus), disease18. While the principle infective agent in Australia appears to and that black-pigmented, anaerobic bacteria, belonging to the be F. necrophorum, other organisms appear to play a role in this genera Porphyromonas and Prevotella, are part of the microbiota5. disease. Recent work has shown that F. necrophorum sub-species Earlier studiesusing culture dependentmethods, showed that in the necrophorum is associated with organisms resembling Porphyro- normal oral microbiota of macropods, Gram-negative anaerobes monas gulae in lumpy jaw in macropods in South Australian zoos19. were poorly represented11. In contrast, Dent and co-workers12 Porphyromonas organisms distinct from both P. gingivalis and reported that macropod oral cavities had a noticeable predomi- P. gulae have been proposed as a novel species14 and were isolated nance of Gram-negative and Gram-positive rods with the facultative with increasing frequency from the oral cavity of macropods in our anaerobic Gram-negative rods comprising 40% of the cultivable studies, which warrants further evaluation into the role of this newly organisms although no Bacteroides spp. were isolated. In a study of described organism in jaw disease. 10 species of kangaroos and wallabies, black-pigmented anaerobic A study of the tammar wallaby pouch young suggested factors that bacteria comprised 21% of their normal oral microbiota13. Thus the protect young animals against potentially pathogenic microbial early culture-based studies have shown variability in the microbes infections could include the microbiome from the maternal saliva20. isolated and suggest that this approach has limitations. The microbiomes of the pouch and saliva from the mother were Koalas do present with severe periodontal disease4 and with severe compared with the gastrointestinal tract (GIT) microbiome of the loss of alveolar bone associated with age and conditions such as food pouch young using 16S rRNA gene comparative methods. Each impaction3 (Figure 1). In koalas <7 years old with good oral health, study site had a unique microbiome20. The maternal pouch har- there was an absence of black-pigmented bacteria, compared to boured 41 unique Actinobacteria phylotypes, while in the saliva koalas >7 years of age, where 50% harboured black-pigmented there were 48 unique Proteobacteria phylotypes. The GIT of the bacteria, the majority of which were identified as Porphyromonas pouch young had a complex microbiome of 53 unique phylotypes gingivalis-like14. Current work characterising this bacterium, shows and of these, only nine were detected at either maternal site. Overall, the organism to be novel and that it may be associated with the majority of bacteria detected were novel species and each study periodontal disease in marsupials [Bird et al, 2015, submitted]. site possessed its own unique microbiome20. Another intriguing question relatesto the koala’s diet which consists In conclusion, a number of culture-based studies on the oral of Eucalyptus leaves. How have the oral bacteria evolved in the microbiota of marsupials were conducted some 20–30 years ago. presence of such a toxic diet (e.g. high in essential oils) and how Now with the deep sequencing culture-independent methods it will does this affect the koala’s oral microbiome? be possible to detect novel bacterial species in their oral cavities Oral necrobacillosis or lumpy jaw as it is commonly known, is a and these are likely to have unique properties. In addition, new leading cause of mortality in captive macropods and has been studies will undoubtedly lead to insights into their evolution, reported in free-ranging macropods15. The disease progresses from diversity and ecological role. We can speculate that co-evolution plaque formation, gingivitis and periodontal disease to a necrotis- of the marsupial oral microbiome has occurred with its host and ing, fatal osteomyelitis,16 (Figure 2) with all macropods susceptible, organisms such as the newly identified porphyromonad unique particularly Eastern grey kangaroos17. Early studies of macropods to marsupials may represent an ancestral lineage distinct from with lumpy jaw showed that Fusobacterium necrophorum was the P. gulae and P. gingivalis. The oral microbiome of our marsupials most frequent isolate from lesions (81% prevalence) as well as the has received little attention and therefore definitely warrants more

Figure 1. Oral disease in the koala presented with an old mandibular fracture with compaction vegetation resulting in bone loss (credit: Ms Lyndall M Pettett).

30 MICROBIOLOGY AUSTRALIA * MARCH 2015 Under the Microscope

Figure 2. Wallaby skull showing swollen lower jaw due to oral necrobacillosis or lumpy jaw. thorough exploration by keen and dedicated microbiologists for 16. Bakal-Weiss, M. et al. (2010) Use of a sustained release chlorhexidine varnish novel bacteria and associated oral diseases. as treatment of oral necrobacillosis in Macropus spp. J. Zoo Wildl. Med. 41, 371–373. doi:10.1638/2010-0004.1 17. Butler, R. (1981) Epidemiology and management of ‘lumpy jaw’ in macropods. References In Fourth International Conference on the Wildlife Diseases Association. Sydney, Australia. fi 1. Tsang, K.L. et al. (2005) Caries and periodontal disease: two diseases, one bio lm. 18. Samuel, J.L. (1983) Jaw disease in macropod marsupials: bacterial flora isolated 23,110–112. Microbiol. Aust. from lesions and from the mouths of affected animals. Vet. Microbiol. 8,373–387. 2. Marsh, P.D. (2003) Are dental diseases examples of ecological catastrophes? doi:10.1016/0378-1135(83)90050-0 149, 279–294. doi:10.1099/mic.0.26082-0 Microbiology 19. Antiabong, J.F. et al. (2013) A molecular survey of a captive wallaby population for 3. Lee, E.F. et al. (2011) Loss of tooth-supporting bone in the koala (Phascolarctos periodontopathogens and the co-incidence of Fusobacterium necrophorum cinereus) with age. Aust. J. Zool. 59,49–53. doi:10.1071/ZO10047 subspecies necrophorum with periodontal diseases. Vet. Microbiol. 163, – 4. Pettett, L.M. et al. (2012) The development of an oral health charting system for 335 343. doi:10.1016/j.vetmic.2013.01.012 koalas (Phascolarctos cinereus). J. Vet. Dent. 29, 232–241. 20. Chhour, K.L. et al. (2010) An observational study of the microbiome of the 5. Bird, P.S. et al. (2002) Oral disease in animals: the Australian perspective. Isolation maternal pouch and saliva of the tammar wallaby, Macropus eugenii, and of – and characterisation of black-pigmented bacteria from the oral cavity of marsu- the gastrointestinal tract of the pouch young. Microbiology 156,798808. pials. Anaerobe 8,79–87. doi:10.1006/anae.2002.0412 doi:10.1099/mic.0.031997-0 6. Pérez-Chaparro, P.J. et al. (2014) Newly identified pathogens associated with periodontitis: A systematic review. J. Dent. Res. 93, 846–858. doi:10.1177/ 0022034514542468 Biographies 7. Sturgeon, A. et al. (2014) Characterization of the oral microbiota of healthy Dr Philip Bird is an Adjunct Associate Professor at the School of cats using next-generation sequencing. Vet. J. 201,223–229. doi:10.1016/ Veterinary Science. His research interests are the oral microbiology j.tvjl.2014.01.024 of human and animals, especially that of native Australian animals. 8. Sturgeon, A. et al. (2013) Metagenomic analysis of the canine oral cavity as revealed by high-throughput pyrosequencing of the 16S rRNA gene. Vet. Micro- Dr Wayne Boardman is Senior Lecturer, at the School of Animal – biol. 162,891 898. doi:10.1016/j.vetmic.2012.11.018 and Veterinary Sciences, University of Adelaide. An experienced 9. Dewhirst, F.E. et al. (2010) The human oral microbiome. J. Bacteriol. 192, wildlife veterinarian, he is a diplomate of the European College of 5002–5017. doi:10.1128/JB.00542-10 Zoological Medicine in Wildlife Population Health. 10. Antiabong, J.F. et al. (2013) The oral microbial community of gingivitis and lumpy jaw in captive macropods. Res. Vet. Sci. 95, 996–1005. doi:10.1016/ Dr Darren Trott is Associate Professor of Veterinary Microbiology j.rvsc.2013.08.010 at The University of Adelaide. His research interests cover compar- 11. Beighton, D. and Miller, W.A. (1977) A microbiological study of normal flora of macropod dental plaque. J. Dent. Res. 56, 995–1000. doi:10.1177/002203 ative aspects of antimicrobial resistance in animals and humans, new 45770560083101 drug development and gastrointestinal microbial ecology.

12. Dent, V.E. (1979) The bacteriology of dental plaque from a variety of zoo- Linda L Blackall is a microbial ecologist who has studied many maintained mammalian species. Arch. Oral Biol. 24, 277–282. doi:10.1016/ 0003-9969(79)90089-X different complex microbial communities ranging from host asso- 13. Samuel, J.L. (1982) The normal ﬂora of the mouths of macropods (Marsupialia: ciated through to free living in numerous environments. Her macropodidae). Arch. Oral Biol. 27, 141–146. doi:10.1016/0003-9969(82)90134-0 research has covered mammalian microbiomes spanning marsu- 14. Mikkelsen, D. et al. (2008) Phylogenetic analysis of Porphyromonas species pials, humans, ruminants and horses and the methods used allow isolated from the oral cavity of Australian marsupials. Environ. Microbiol. 10, elucidation of massive microbial complexity and function in these 2425–2432. doi:10.1111/j.1462-2920.2008.01668.x diverse biomes. She is a Professor of Biosciences at Swinburne 15. Borland, D. et al. (2012) Oral necrobacillosis (‘lumpy jaw’) in a free-ranging population of eastern grey kangaroos (Macropus giganteus) in Victoria. Aust. University of Technology in the Faculty of Science, Engineering and Mammal. 34,29–35. doi:10.1071/AM10031 Technology.

MICROBIOLOGY AUSTRALIA * MARCH 2015 31 Lab Report

Relative abundance of Mycobacterium in ovine Johne’s disease

Andy O LeuA, Paul PavliB,C, David M GordonA, Jeff CaveD, Jacek M GowzdzE, Nick LindenD, E A,C A,B,C,F Grant Rawlin , Gwen E Allison and Claire L O’Brien AResearch School of Biology, Australian National University, Acton, ACT 0200, Australia BGastroenterology and Hepatology Unit, Canberra Hospital, Garran, ACT 2605, Australia CMedical School, Australian National University, Acton, ACT 0200, Australia DDepartment of Environment and Primary Industries, Wodonga, Vic. 3690, Australia EAgriBio, Bundoora, Vic. 3083, Australia FCorresponding author. Tel: +61 2 6244 4023, Email: [email protected]

No study has determined what proportion of the total micro- 38%) of the total microbiota of samples from sheep with biota comprises the genus Mycobacterium in ovine Johne’s OJD, and 0-85% (average 13%) of sheep without OJD. Only disease (OJD) tissues. We aimed to assess the relative abun- sheep with OJD had samples that were positive for the IS900 dance of Mycobacterium in the ileocaecal lymph node, (MAP) element. Mycobacterial strains other than MAP may involved and uninvolved ileal mucosa from sheep with and provide competitive exclusion of MAP and should be further without OJD, using three extraction methods. Eight sheep, investigated. four with and four without OJD, were recruited. Pyrosequen- cing of the 16Sr RNA gene amplicons for all samples revealed Mycobacterium avium subspecies paratuberculosis (MAP)is that Mycobacterium represented between 0-92% (average the etiologic agent of ovine Johne’s disease (OJD), a chronic,

32 10.1071/MA15010 MICROBIOLOGY AUSTRALIA * MARCH 2015 Lab Report

contagious granulomatous enteritis of ruminants. The disease causes signiﬁcant morbidity and mortality worldwide, resulting in signiﬁcant economic losses1.

To our knowledge, no study has determined what fraction of the microbial community of non-OJD and OJD affected tissue comprises Mycobacterium. Cheung et al. (2013)2 found that Mycobac- terium tuberculosis represented less than 1% of the total microbial community of sputum samples of infected patients. Does Myco- bacterium comprise such a small fraction of the microbial community in all infectious states and specimens? MAP is a putative trigger of Crohn’s disease in humans, and is detected in some studies but not others, including a recent study of ours3. Is this because it is not being detected? Figure 1. Ovine Johne’s disease affected sheep #2, showing physical signs of wasting. While other studies have quantified Mycobacterium or MAP in 4,5 clinical and subclinical specimens , no study has determined what having OJD, or not, based on physical signs, including condition, fraction of the microbial community comprises Mycobacterium in scouring, ill-thrift, and lethargy during mobility. Diagnosis was mucosa from sheep with and without OJD. The primary aim is to confirmed by a NATA-accredited pathology laboratory (AgriBio, assess the relative abundance of Mycobacterium in the microbial Victoria), and included MAP culture and DNA analysis, as well as communities of macroscopically normal bowel mucosa obtained microscopic visualization of acid-fast bacteria using ZN staining . >50 cm downstream the ileocaecal valve (sheep without OJD) Sheep 1 was initially suspected to have OJD, however negative or involved mucosa (sheep with OJD), involved (or normal for culture and staining results excluded it from a diagnosis of OJD, and sheep without OJD) ileal mucosa adjacent the ileocaecal lymph it was found to have metastatic cancer. node, and the ileocaecal lymph node of sheep with and without Pyrosequencing showed that Mycobacterium represented between OJD. 0–92% (average 38%) of the total microbiota of samples from sheep Samples of involved and uninvolved ileal mucosa, and ileocaecal with OJD, and 0-85%(average 13%) ofsheep without OJD (Figure 2). lymph nodes were collected from eight sheep, four with and four Mucosal samples from sheep 7 were excluded due to the inability to without OJD, from farms around Rutherglen, Australia. Sterile amplify products from them. Sheep 4 and 5 each had a positive gloves and implements were used to extract the ileocaecal node lymph node sample for one extraction, comprising 6% and 68% before the bowel was opened, so that it did not come into contact Mycobacterium reads, respectively. These levels were similar tothat with the skin or bowel microbiota. Total DNA was extracted from observed in the mucosal samples from the same animal. each sample using three different extraction methods. The first The lymph node and mucosa samples of sheep 1 were negative protocol followed the Qiagen DNeasy kit protocol and included an when tested with the MAP-specific primers. Sheep 1 may have had a enzymatic lysis step and bead-beating step. The second and third paucibacillary form of OJD. Samples from all other sheep with extractions were the same as the first, except the second protocol OJD were positive, except the normal mucosa sample of sheep 4 included a freeze-thaw step, and the third protocol a boiling step. (Table 1). All samples from sheep without OJD were negative for the Technical replicates were included for the first protocol. All samples IS900 element. were amplified using barcoded primers targeting the universal bacterial16S rRNA gene, and pyrosequenced using a 454 Genome No significant effect of extraction method was detected for either Sequencer FLX-Titanium platform, as previously described3. Simpson or Shannon diversity estimates (Simpson Diversity, AOV: Sequences were analysed using Mothur6. IS900 PCR was used to P > F = 0.384; Shannon Diversity, AOV: P > F = 0.443), nor technical determine whether or not Mycobacterium detected in a given replicates (Simpson Diversity, AOV: P > F = 0.398; Shannon Diver- sample was MAP, or not. sity, AOV: P > F = 0.566).

Four sheep (1, 2, 4 and 5; for example, see Figure 1) were diagnosed Each sample was covered by an average of 2, 644 quality sequences. with OJD by qualiﬁed veterinarians and pathologists, using methods Six phyla (Firmicutes, Bacteroidetes, Actinobacteria, Proteobac- previously described7,8. Brieﬂy, sheep were initially assessed as teria, Synergistetes, and TM7) represented ~98% of all sequences

MICROBIOLOGY AUSTRALIA * MARCH 2015 33 Lab Report

100

70 (%) 60

30 Mycobacterium 20

Figure 2. Proportion of involved and normal mucosa and lymph node tissues (sheep 4 and 5) containing Mycobacterium. Sheep with Johne’s disease are coloured orange, without blue. Technical replicates (normal mucosa only) have diagonal texturing. The ﬁrst numeral in the label indicates the animal number; NM, normal mucosa; IM, involved mucosa; LN, lymph node; the second numeral indicates the DNA extraction: ‘1’, ‘2’ and ‘3’ are replicates, ‘12’ is the technical replicate for normal mucosa (DNA extraction 1) only. Sheep 1, 2, 4 and 5 had OJD, sheep 3, 6 and 8 did not.

Table 1. Sheep characteristics, pathology and MAP-speciﬁc PCR results Sheep 1 2 3 4 5 6 7 8

Diagnosis OJD and MIC OJD Non-OJD OJD OJD Non-OJD Non-OJD Non-OJD

SexFFFFFFFF

Age74774636

Vaccination N Y N N Y N Y N

Breed Merino Merino Merino X Merino X Merino Merino Merino Merino Leicester Leicester

Intestinal Severe Moderate None Moderate Mild None None None lesions

MAP culture + + – + + – – –

AFB in Absent Occasional Absent Numerous Numerous Absent Absent Absent Involved Mucosa

AFB in Absent Absent Absent Moderate Numerous Absent Absent Absent Lymph node

MAP-speciﬁc IS900 PCR

NM–+––+–––

IM–+–++–––

LN–+–++–––

OJD, ovine Johne’s disease; MIC, metastatic intestinal cancer; MAP, Mycobacterium avium subspecies paratuberculosis; Y, yes; N, no; AFB, acid-fast bacteria; NM, normal mucosa; IM, involved mucosa. All samples were positive for bacterial DNA using the 16S rRNA gene primers, except sheep 7 samples.

(Figure 3). Actinobacteria, of which Mycobacterium is a member, To our knowledge, this is the ﬁrst study to describe the full was overrepresented in sheep with OJD, except sheep 1. Microbial microbial community of mucosa from sheep with and without community diversity declined as Mycobacterium increased OJD. Although studies have enumerated MAP in the feces of sheep (R2=0.705, p=0.001) (Figure 4). using RT-PCR5,9, no study has determined what fraction of the

34 MICROBIOLOGY AUSTRALIA * MARCH 2015 Lab Report

Figure 3. Proportion of the six dominant bacterial phyla in tissues from sheep with and without ovine Johne’s disease (OJD), showing a predominance of Actinobacteria, of which Mycobacterium is a member, in sheep with OJD. The ﬁrst numeral in the label indicates the animal number; NM, normal mucosa; IM, involved mucosa; LN, lymph node; the second numeral indicates the DNA extraction: ‘1’, ‘2’ and ‘3’ are replicates, ‘12’ is the technical replicate for normal mucosa only. Sheep 1, 2, 4 and 5 had OJD, sheep 3, 6 and 8 did not.

Samples from sheep without OJD were not positive for MAP 7 Sheep 1 culture or the IS900 element, but other strains of Mycobacterium Sheep 2 6 were present, representing on average 13% of the total microbiota. Sheep 3 5 Strains of Mycobacterium other than MAP could occupy a niche Sheep 4 in the sheep gastrointestinal tract that MAP would otherwise 4 Sheep 5 occupy, thereby protecting the animal from colonization of 3 Sheep 6 MAP. Future studies should assess the diversity of Mycobacterium Sheep 8 Shannon index 2 strains associated with the mucosa of sheep with and without

1 OJD.

0 We found the abundance of Mycobacterium, relative to other 0 20406080100120 bacterial taxa in the mucosal samples, was significantly higher in Mycobacterium (%) sheep with OJD. Increases in Mycobacterium result in large Figure 4. Figure showing a negative correlation between the Shannon diversity index and the proportion of Mycobacterium in the mucosal decreases in Firmicutes. Members of the Firmicutes are important microbiota of sheep. Sheep 1, 2, 4 and 5 were diagnosed with OJD. producers of short-chain fatty acids, which are shown to improve 10 microbial community Mycobacterium comprises, and whether or intestinal barrier function and repress inflammation . Dysbiosis not these proportions differ in sheep with and without OJD, as and reduced diversity would likely exacerbate symptoms of OJD, well as various tissue types. as beneficial bacteria are decreased and their roles in homeostasis unfulfilled. Bacterial translocation to the ileocaecal lymph node was observed in 50% (2/4) of sheep diagnosed with OJD. The microbial community Mycobacterium makes up a large fraction of the microbial com- of these nodes was similar to that of the involved and normal mucosa munities of mucosa from sheep with OJD. Future studies should from the same sheep. Bacteria in the node are likely to be secondary determine if MAP is the sole contributor to these levels of Myco- invaders, present in the node due to a breakdown in the mucosal bacterium in sheep with OJD, and assess the diversity of Mycobac- barrier. Using the same methods described here, we found that terium in sheep with and without OJD. Other Mycobacterium bacterial translocation in Crohn’s disease of humans is also non- strains may provide competitive exclusion of MAP. specific. We also did not detect Mycobacterium in this study using 16S rRNA gene sequencing3. This suggests that Mycobacterium is Acknowledgements unlikely to be a cause of Crohn’s disease. We thank Greg Seymour for assisting in the collection of specimens.

MICROBIOLOGY AUSTRALIA * MARCH 2015 35 Lab Report

References the interaction between the innate inflammatory response and the 1. Salem, M. et al. (2013) Mycobacterium avium subspecies paratuberculosis: intestinal microbiome. an insidious problem for the ruminant industry. Trop. Anim. Health Prod. 45, 351–366. doi:10.1007/s11250-012-0274-2 David Gordon received his BSc from King’s College London and 2. Cheung, M.K. et al. (2013) Sputum microbiota in tuberculosis as revealed by 16S rRNA pyrosequencing. PLoS ONE 8, e54574. doi:10.1371/journal.pone.0054574 his PhD from McGill University, Montreal. His research concerns the 3. O’Brien, C.L. et al. (2014) Detection of bacterial DNA in lymph nodes of Crohn’s ecology, population genetics and evolution of the Enterobacter- – disease patients using high throughput sequencing. Gut 63, 1596 1606. iaceae, especially E. coli. doi:10.1136/gutjnl-2013-305320 4. Park, K.T. et al. (2014) Development of a novel DNA extraction method for Jeff Cave graduated from Murdoch University in 1988 and spent identification and quantification of Mycobacterium avium subsp. paratuberculosis from tissue samples by real-time PCR. J. Microbiol. Methods 99,58–65. two years in mixed private practice in South Australia before working doi:10.1016/j.mimet.2014.02.003 in the Pacific for much of the 1990s. For the past 16 years he has 5. Logar, K. et al. (2012) Evaluation of combined high-efficiency DNA extraction and fi real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in worked as the District Veterinary Of cer in Wodonga, Victoria. subclinically infected dairy cattle: comparison with faecal culture, milk real-time PCR and milk ELISA. BMC Vet. Res. 8, 49. doi:10.1186/1746-6148-8-49 Jacek Gwozdz is a Veterinary Pathologist at the State veterinary 6. Schloss, P.D. et al. (2009) Introducing mothur: open-source, platform-indepen- laboratory in Victoria. He received his PhD in Veterinary Science dent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537–7541. doi:10.1128/ from the Massey University in New Zealand and worked over the past AEM.01541-09 16 years in research and diagnostic microbiology and pathology. 7. Gwozdz, J.M. et al. (2000) Vaccination against paratuberculosis of lambs already infected experimentally with Mycobacterium avium subspecies paratubercu- NickLinden is a research scientist with the Victorian Department of losis. Aust. Vet. J. 78,560–566. doi:10.1111/j.1751-0813.2000.tb11902.x 8. Whittington, R.J. et al. (2013) Development and validation of a liquid medium Primary Industries and Environment, he has a particular interest in (M7H9C) for routine culture of Mycobacterium avium subsp. paratuberculosis the lamb supply chain and is passionate about linking on farm to replace modified Bactec 12B medium. J. Clin. Microbiol. 51, 3993–4000. doi:10.1128/JCM.01373-13 production through to carcass appraisal and consumer acceptance. 9. Sting, R. et al. (2014) Detection of Mycobacterium avium subsp. paratubercu- His recent projects have been focused on lamb feed conversion losis in faeces using different procedures of pre-treatment for real-time PCR in efficiency as well as on-line grading of lamb carcasses. comparison to culture. Vet. J. 199,138–142. doi:10.1016/j.tvjl.2013.08.033 10. Pryde, S.E. et al. (2002) The microbiology of butyrate formation in the human Grant Rawlin is Research Leader of Veterinary Pathobiology at the colon. FEMS Microbiol. Lett. 217, 133–139. doi:10.1111/j.1574-6968.2002. tb11467.x State veterinary laboratory in Victoria. His career has spanned veterinary diagnostics as a pathologist, research in veterinary and human microbiologyand research intocontrol of infectiousdiseases Biographies of animals at a population level. Andy Leu graduated from the Australian National University (ANU) with a Bachelor of Medical Science (Honours I) in 2013. His Honours Dr Gwen Allison received her PhD in Microbiology from North project examined the relative abundance of Mycobacterium avium. Carolina State University. Her research interests have included paratuberculosis (MAP) in Johne’s disease tissues. Andy was re- molecular biology of lactic acid bacteria and microbial ecology of cently awarded an Australian Postgraduate Award scholarship to the gastrointestinal tract and environment. The current research pursue a PhD under the supervision of Dr Gene Tyson. was conducted when Dr Allison was a Senior Lecturer at the ANU.

Paul Pavli is a gastroenterologist with clinical and basic scientiﬁc Dr Claire O’Brien received her PhD from the Australian National research interests in the inﬂammatory bowel diseases. He was on University Medical School (ANUMS) in 2012. Her main research the steering committee of the multicentre Australian study using interest is the gut microbiome, particularly in Crohn’sdisease. Claire long-term anti-mycobacterial agents in Crohn’s disease (Selby is an NHMRC Peter Doherty Fellow at the ANUMS and Canberra et al., Gastroenterology, 2007). Current research interests are in Hospital.

36 MICROBIOLOGY AUSTRALIA * MARCH 2015 ASM Affairs

Interactions with other microbiology societies through Microbiology Australia

The September 2014 issue of Microbiology Australia was a special In addition, the articles remind us that much is owed to the Turks issue that reflected on the ‘Microbial diseases and products that for the early lessons they learned through their contributions to shaped world history’. A major focus involved microbes of WWI infection control. This is particularly so for their early adoption of and their effects on ANZACs and Turkish soldiers. The timing of variolation for the prevention of smallpox, an experience that led the issue is particularly fitting as we reflect on the 100th anniversary directly to Jenner’s seminal work with cowpox and the development of the terrible losses suffered by both sides involved in the Gallipoli of modern vaccination. conflict. At the invitation of the Turkish Microbiology Society, Professor Paul Young, President of ASM, Ian Macreadie, Editor of Microbiology Australia and Ipek Kurtböke attended the annual Turkish Microbiology Society meeting in Antalya, Turkey in November 2014. They participated in the opening ceremony and gave scientific presentations during the meeting. At the opening ceremony, Professor Young reminded the audience of the strong bonds that were forged between our three countries after WWI and which remain to this day. This connection was most notably encapsulated in the words of Ataturk in 1934. Professor Young’s quotation of this very moving tribute gained very warm applause from the audience: Those heroes that shed their blood and lost their lives...You are now lying in the soil of a friendly country. Therefore rest in peace. There is no difference between the Johnnies and the Mehmets to us where they lie side by side here in this country of ours...You, the mothers, who sent their sons from faraway _ Nezahat Gu¨ rler, Paul Young, Ian Macreadie, Ipek Kurtbo¨ ke, Philippe countries wipe away your tears; your sons are now lying in Desmeth and Bu¨ lent Gu¨ rler. our bosom and are in peace, after having lost their lives on this land they have become our sons as well. The idea for the issue came from Ipek Kurtböke who Guest Edited the joint effort between the Australian, New Zealand and We have learned much from our interactions. It was a privilege to be Turkish Microbiology Societies. The issue contains excellent hosted by such gracious hosts and we look forward to welcoming a articles on the major diseases of world history, and early efforts to delegation of Turkish microbiologists to Canberra for ASM2015. control them. The success of our interactions with other microbiology societies The Turkish articles were highly informative about their experiences is something ASM would like to foster and it is hoped that every with smallpox, tuberculosis and typhus as well as the huge losses few years a similar joint issue of Microbiology Australia can be the Turkish troops suffered at the hands of infectious diseases. produced.

ASM History SIG: Microbiology Australia

The ASM History SIG was launched at ASM Adelaide 2013 at a institutions). In future it is intended to have a History of Australian meeting on 12 noon Tuesday 9th July 2013 and Dr Diane Lightfoot Microbiology Symposium at the ASM annual scientific meeting. was elected convener for the first 2 years of this SIG. The AGM of the History SIG will be held at the ASM scientific The purpose of this SIG is to record the history of Microbiology in meeting in Canberra July 2015. Australia, in the ASM archives, including details about certain If ASM members have that significant ASM memorabilia that they microbiologists, microbiological institutions, and microorganisms, would like to donate to the ASM archives or suggestions of topics as well as disseminate this information to ASM members. suitable for possible symposia at the ASM Annual Meetings. Please send details of memorabilia or suggestions/topics of interest or for Regularly notes on topics of interest will be contributed to the ASM possible symposia to: Victorian Branch Newsletter from the History SIG (e.g. prominent microbiologists, significant microbiological discoveries by Victorian History SIG convener, c/o Australian Society for Microbiology Office, microbiologists, microbiological institutions/major events at these 9/397 Smith Street, Fitzroy, Vic. 3068, Australia.

MICROBIOLOGY AUSTRALIA * MARCH 2015 10.1071/MA15011 37 ASM Affairs

Recent developments in virology by Australian researchers

Joseph R Freitas and Suresh Mahalingam Institute for Glycomics, Grifﬁth University, Gold Coast, Queensland

Virology is a growing field within Australia. Increased funding is collaborative input from University of Melbourne researchers merits being allocated to discovering how viruses interact with their its mention here. host/vectors(s) and to the development of better treatments Viruses with the ability to pass from animals to humans (‘zoonotic and vaccines. There have been many recent exciting new develop- viruses’) are very unpredictable because they have the potential to ments in Australian virology. Space limitations mean that we can mutate and become deadly in new hosts. The most notable local only highlight a small number of these achievements in this brief example of this is the Hendra virus outbreaks that occurred over the overview of the current virology landscape in Australia. past two decades. Research into this virus and its natural hosts led to The increasing prevalence or re-emergence of certain alphaviruses, the successful development and deployment of a vaccine against 4 fi such as Ross River virus (RRV), Sindbis virus, and Chikungunya Hendra in 2012 . A newly identi ed virus found in Australian bats, 5 virus, is a cause for concern and has attracted increased research dubbed Cedar virus, by Marsh et al. in 2012 was found to be very interest in recent years. These viruses are known as arthritogenic similar to the Hendra and Nipah viruses. The one key difference is alphaviruses because they cause arthritis-like symptoms. The ability that it does not cause disease in several mammals that are suscep- of these viruses to directly induce bone pathology has remained tible to Hendra and Nipah. This fascinating discovery will no doubt poorly defined to date. Chen et al.1 recently shed some light on this assist scientists to understand what exactly it is that makes certain issue by revealing that RRV can infect human osteoblasts and that viruses deadly and others less so. osteoblast infection leads to IL-6-dependent bone loss in a mouse Norovirus (NoV) is the leading cause of gastroenteritis worldwide model. This discovery of the interaction between osteoblasts, and is known to cause thousands of deaths in developing countries. inflammatory factors and alphaviruses is a plausible explanation for Most outbreaks occur within institutional settings such as hospitals how viruses cause chronic joint pain. and aged-care facilities. One particular genotype of the virus is responsible for the majority of infections, the genogroup II geno- Infections with certain viruses, such as West Nile virus (WNV) and type 46. Since current treatment for NoV infections is largely dengue virus, can lead to encephalitis, which is associated with preventative, there is an urgent need for an effective vaccine or high mortality. Inflammatory cells play a central in the progression antiviral. By using high throughput screening methods against the of these diseases. Currently there are no specific targeted treat- RNA polymerase of NoV, Eltahla et al.7 identified a very promising ments to modulate the action of inflammatory cells. Getts et al.2 target for development of effective antivirals against this disease. recently demonstrated that infusion of immune-modifying microparticles (IMPs) into WNV-infected mice significantly reduced With these and the many other researchers around Australia cur- fi the symptoms of central nervous system infection. The continued rently active in the eld, we can expect continuing major advances in injection of IMPs to mice over several days led to an improved the discipline of virology in 2015 and in the years to come. survival rate. Similar therapeutic effects were shown for other References inflammation-mediated diseases. The therapeutic potential of 1. Chen, W. et al. (2014) Arthritogenic alphaviral infection perturbs osteoblast such IMPs for a variety of immune-related disorders looks to be function and triggers pathologic bone loss. Proc. Natl. Acad. Sci. USA 111, very promising. 6040–6045. doi:10.1073/pnas.1318859111 2. Getts, DR et al. (2014) Therapeutic inflammatory monocyte modulation using Dengue (DENV) is a mosquito-borne virus that infects hundreds immune-modifying microparticles. Sci. Transl. Med. 6, 219ra7. of millions of people annually. It has a widespread geographical 3. Dejnirattisai, W. et al. (2015) A new class of highly potent, broadly neutralizing prevalence that continues to expand. There is an urgent need for an antibodies isolated from viremic patients infected with dengue virus. Nat. Immu- nol. 16, 170–177. doi:10.1038/ni.3058 effective vaccine against DENV. However, this has proven difficult 4. Broder, C.C. et al. (2013) A treatment for and vaccine against the deadly Hendra and due to the virus having four different serotypes, with a general Nipah viruses. Antiviral Res. 100,8–13. doi:10.1016/j.antiviral.2013.06.012 consensus that a successful vaccine will need to induce immunity to 5. Marsh, G.A. et al. (2012) Cedar virus: a novel Henipavirus isolated from Australian each specific serotype. The identification of antibodies with broad bats. PLoS Pathog. 8, e1002836. doi:10.1371/journal.ppat.1002836 cross-reactivity to all serotypes by Dejnirattisai et al.3 led to the 6. Eden, J.S. et al. (2013) Recombination within the pandemic Norovirus GII.4 lineage. J. Virol. 87,6270–6282. doi:10.1128/JVI.03464-12 discovery of a novel DENV epitope, which has high potential for use 7. Eltahla, A.A. et al. (2014) Nonnucleoside inhibitors of Norovirus RNA as a successful vaccine antigen. Although this study was performed polymerase: scaffolds for rational drug design. Antimicrob. Agents Chemother. outside of Australia, the threat of DENV in Australia and the strong 58, 3115–3123. doi:10.1128/AAC.02799-13

38 10.1071/MA15013 MICROBIOLOGY AUSTRALIA * MARCH 2015 ASM Affairs

Clinical Serology and Molecular SIG

From the retiring National Convenor, David Dickeson

The Clinical Serology Special Interest Group was established after a attended, reports were tabled for NSW, QLD, VIC, SA and WA, and a meeting in Adelaide in 1989 with Peter Robertson (now retired from call for nominations for new national convenor, secretary and Prince ofWales Hospital,Randwick) the inaugural national convenor workshop organiser was made. and only a few dozen members. It was established to provide a forum After over 10 years of service as national convenor and serving as for the discussion of serological techniques with the objective to secretary/treasurer of the NSW branch since 1993, I will be handing develop a strategy to monitor and control the quality of diagnostic over the duties and responsibilities of the ASM CS&M SIG to the reagents. This was published in 1994 (Backhouse et al., Australian new convenor. Coordinating, participating and helping with all the Microbiologist,1994;15:37–45).BranchesofthisSIGwereformedin events and discussions of this SIG has been a pleasure for over most states from 1989 onwards. I became involved as NSW branch 22 years. I would like to thank Wayne for his help and advice as treasurer in 1993 with Leon Heron as convenor and I have remained secretary and administrator of the SERSIG email routing and espe- as secretary/treasurer ever since. The group was expanded in 2003 cially thank Sheena for being the driving force behind the working to include molecular techniques in the medical diagnostic industry group, organising teleconferences, emails to members and letters and the combined SIG convenor was Robyn Wood (formerly from to governing bodies. I trust the Clinical Serology and Molecular Queensland Medical Laboratory, then TGA). In 2004 I took on the SIG will continue to meet the needs of members by providing position as convenor at the ASM annual meeting in Sydney and have useful and timely scientific information about diagnostic serology continued in that role until now. and molecular techniques and related matters. I am sure the SIG The CS&M SIG Working Group was formed in 2009 after several will be left in good hands with the help of the new executive. ASM meetings and NRL workshops with the great assistance of New office bearers for the SIG are: Sheena Adamson (Communicable Diseases Branch, NSW Health) Convenor: Linda Hueston, Pathology West – ICPMR, Westmead, who remains as the working group secretary. This group is NSW. overseen by the ASM Clinical Microbiology Standing Committee Email: [email protected] and aims to be a proactive source of education and information and able to give advice on serology and related molecular testing. Secretary: Megan Wieringa, Monash Health, Clayton, Victoria. Teleconferences held every 3 months have kept an expert group of Email: [email protected] scientists and medical microbiologists in contact. Dissemination Workshop organiser: Bruce Wong, PaLMS, RNSH, St Leonards, NSW. of information has been achieved through ASM, NRL, RCPA QAP, Email: [email protected] various direct emails and the national email routing ‘SERSIG’, which links all members with requests for information or help in State Branch Convenors 2014 resolving problems. Now SERSIG has 419 members. NSW: Deane Byers, RCPA. Email: [email protected] At the ASM annual scientific meeting each year, we organise a workshop with topics including QAP, case studies and issues with Victoria: Wayne Dimech (retiring), NRL. diagnostic kit failures or problems. Previous workshops connected Email: [email protected] with ASM annual meetings in Adelaide, Melbourne and Sydney were Queensland: Cheryl Bletchley and Theo Sloots (retiring). organised by Ros Escott (RCPA QAP now retired). In 2014 a molec- Email: [email protected] ular diagnosis workshop was held on the Saturday afternoon at the Peter Doherty Institute prior to the national meeting. The meeting South Australia: Trish Hahesy, IMVS. was chaired by Wayne Dimech and Darren Jardine with over 80 Email: [email protected] attending. It included topics on molecular testing, validation and Western Australia: Justin Morgan, PathWest Laboratory standards, choosing equipment and new technologies. Medicine WA. Email: [email protected] An Annual General Meeting of the SIG is organised by the convenor at each yearly meeting and it includes branch reports, industry input Tasmania: Louise Cooley, RHH. and discussion of current topics. In Melbourne last year 17 members Email: [email protected]

MICROBIOLOGY AUSTRALIA * MARCH 2015 10.1071/MA15014 39 ASM Affairs

Report from the ASM Antimicrobial Special Interest Group (ASIG)

The BioMerieux Identifying Resistance Award in 2014 went to A/Prof Denis Spelman for his long continued support for antimicrobials and infectious control practices; this was presented in Melbourne annual scientiﬁc meeting.

ASIG became affiliated with the International Society of Chemo- John Merlino, on behalf of ASIG therapy (ISC) after a formal invitation. Some of our members who fi ASM ASIG Convenor/Chair specialise in speci c antimicrobial areas have been invited and taken Email: part in symposiums and other events at their own costs. Members of [email protected] ASIG committee actively communicate with other local and international antimicrobial groups on emerging issues in antimicrobial The ASM Antimicrobial Special Interest Group (ASIG) is made up resistance for example with the MRSA International Committee. of colleagues and ASM members you have an interest in antimicro- This remains ongoing in 2014–15. ASIG Members have also been bials. Members of the ASIG committee include medical, veterinary, involved in European Congress Clinical Microbiology and Infectious CDS Users, mycology and parasitology consultants. The Antimicro- Diseases (ECCMID) meetings overseas – expenses met by their own bial SIG is one of the largest Special Interest Groups in the ASM. trust fund not ASM. Members have been invited and involved in In 2012 we had over 923 ASIG members. In 2013 we had 721 writing articles on antimicrobials for Microbiology Australia and members listed under ASIG. Some of our members have retired. overseas journals. When requested ASIG members who specialise Numbers are yet to be finalised in 2014. in specific areas of antimicrobials interests review articles for local and international journals. In 2014 ASM ASIG members were actively involved with local and national ASM Branch meetings in various states – this is usual in the The ASIG convenor/committee and members communicates with form of communication and newsletters. Committee actively com- Division 1 Chairs, informally inviting overseas speakers in organising municates via emails and website www.asig.org.au on emerging symposiums and workshops at ASM Annual Scientific Meetings. issues – methodology on susceptibility testing and standards on ASIG continues to review submissions and comments from other antimicrobials and antimicrobial resistance. Members of the com- Medical and Antimicrobial Groups in regards to antimicrobial issues, mittee actively take part in planning workshops titles and speakers, e.g. Royal College of Pathologists Australasia Advisory Committee, e.g. Annual Scientific ASM Meetings – this has been the focus for AIMS, NATA, NPAAC, with the ASM Clinical Standing Committee many years. Discussions on antimicrobial issues take place at these when required. ASIG continues support of teaching and research meetings. activities where possible by seeking speakers for events. ASIG promotes the advancement of ASM SIG antimicrobials by presenting In Melbourne in 2014, the Antimicrobial Special Interest Group lectures/visits to Universities and Colleges nationally and interna- Workshop on Antimicrobial Resistance focused on MALDI-TOF and tionally as required. Susceptibility Testing with both CLSI and EUCAST methodology. This was a DRY Workshop with practical slide demonstrations with ASIG is involved with the Continuing Education Program, involved discussions and a QUIZ with equipment such as buzzers provide with the AIMS APACE Official Certificate of Attendance is given out via the RCPA. Speakers’ presentations are available on (powerpoint at all ASIG workshops at National meetings. These certificates are in PDF) on the ASIG website www.asig.org.au, http://asig.org. important for professional developments and career advancements au/australian-society-for-microbiology-asm-2014-annual-scientific- in industry. As the convenor I thank all our members and sponsors meeting-melbourne-australia-workshop-presentations/. for their continued support.

40 10.1071/MA15015 MICROBIOLOGY AUSTRALIA * MARCH 2015 12-15 July 2015 QT CANBERRA 1 London Circuit, Canberra

Rubbo Oration Janet Jansson, Pacific Northwest National Laboratory, USA Bazeley Oration Yoshihiro Kawaoka, University Wisconsin -Madison, USA, University Tokyo, Japan Plenary Speakers Chantal Abergel, CNRS-AMU, France Judith Berman, Tel Aviv University, Israel Ed DeLong, MIT, USA Jorge Galan, Yale University, USA Stefan Schwarz, Friedrich-Loeffler-Institut, Germany Public Lecture: From Guts to Great Oceans Janet Jansson, Mike Manefield, Ed DeLong Workshops Antimicrobials Bioinformatics for microbial ecology Imaging in microbiology Methods in microbial proteomics and metabolomics Women in leadership EduCon: Microbiology Educators’ Conference preceding asm2015. Joint Mycology Meeting with the Australasian Mycological Society, July 15-16.

Annual Scientific Meeting and Trade Exhibition [email protected] http://asmmeeting.theasm.org.au/ The highly evolved senses of a hunter

With continuous, random access and no-batch processing the Panther® system, along with our highly sensitive and speciﬁc Aptima® assays, provide on-demand performance for a wide range of infectious diseases.

Gen-Probe Australia Pty Ltd. ABN 91 150 687 773 . Suite 402, Level 4, 2-4 Lyon Park Road, Macquarie Park, NSW, Australia, 2113 Phone: +61 2 9888 8000 Fax: (02) 9870 7555

"%4"64&/ 3FW © 2015 Hologic, Inc. All rights reserved. Hologic, Science of Sure, Aptima, Panther and associated logos are trademarks and/or registered trademarks of Hologic, Inc. and/or its subsidiaries in the United States and/or other countries. This information is intended for medical professionals and other markets and is not intended as a product solicitation or promotion where such activities are prohibited. Because Hologic materials are distributed through websites, eBroadcasts and tradeshows, it is not always possible to control where such materials appear. For specific information on what products are available for sale in a particular country, please contact your local Hologic representative or write to [email protected] *Pending listing on the Australian Register of Therapeutic Goods (ARTG)

Mammalian Microbiomes CONFIDENCE *O.JDSPCJPMPHJDBM*EFOUJGJDBUJPOGSPN$IBSMFT3JWFS"VTUSBMJBBOE/FX;FBMBOE

Mammalian Microbiomes CONFIDENCE O.JDSPCJPMPHJDBMEFOUJGJDBUJPOGSPN$IBSMFT3JWFS"VTUSBMJBBOE/FX;FBMBOE