Mammalian microbiomes - CSIRO Publishing

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Volume 36 Number 1 March 2015Volume 36 Number 1 March 2015

Mammalian microbiomes

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Volume 36 Number 1 March 2015

ContentsVertical Transmission 2

Jon Iredell

Guest Editorial 3Mammalian microbiomes 3

Linda L Blackall

In Focus 4Methane matters in animals and man: from beginning to end 4

Emily Hoedt, Paul Evans, Stuart Denman, Chris McSweeney, Paraic ÓCuív and Mark Morrison

The marine mammal microbiome: current knowledge

and future directions 8

Tiffanie M Nelson, Amy Apprill, Janet Mann, Tracey L Rogers and Mark V Brown

The role of the gut microbiome in host systems 14

Clarissa Febinia, Connie Ha, Chau Le and Andrew Holmes

Under the Microscope 18Modulation 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

Gastrointestinal microbiota, diet and brain functioning 25

Shakuntla Gondalia and Andrew Scholey

Marsupial oral cavity microbiome 29

Philip S Bird, Wayne SJ Boardman, Darren J Trott and Linda L Blackall

Lab Report 32Relative abundance of Mycobacterium in ovine Johne’s disease 32

Andy O Leu, Paul Pavli, David M Gordon, Jeff Cave, Jacek M Gowzdz, Nick Linden, Grant Rawlin, Gwen E Allison and Claire L O’Brien

ASM Affairs 37Interactions with other microbiology societies

through Microbiology Australia 37

ASM History SIG: Microbiology Australia 37

Recent developments in virology by Australian researchers 38

Clinical Serology and Molecular SIG 39

Report from the ASM Antimicrobial Special Interest Group (ASIG) 40

MICROBIOLOGY AUSTRALIA • MARCH 2015 1

Dr Gary Lum Dr John MerlinoProf. Wieland MeyerProf. William RawlinsonDr Paul SelleckDr David SmithMs Helen SmithDr Jack WangDr Paul Young

The Australian Societyfor Microbiology Inc.9/397 Smith StreetFitzroy, Vic. 3065Tel: 1300 656 423Fax: 03 9329 1777Email: [email protected] 24 065 463 274

For Microbiology Australiacorrespondence, see address below.

Editorial teamProf. Ian Macreadie, Mrs Jo Macreadieand Mrs Hayley Macreadie

Editorial BoardDr Chris Burke (Chair)Prof. Mary BartonProf. Linda BlackallProf. Sharon ChenProf. Peter ColoeDr Narelle FeganDr Geoff HoggProf. Jonathan IredellDr I

.pek Kurtböke

Subscription ratesCurrent subscription rates are availablefrom the ASM Melbourne offi ce.

Editorial correspondenceProf. Ian Macreadie/Mrs Jo MacreadieTel: 0402 564 308 (Ian)Email: [email protected]

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ISSN 1324-4272eISSN 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).

Jon Iredell

President of ASM

Along with all the sciences, our discipline is evolving quickly in all

areas from pure research to applied and professional. The ASM was

formed more than half a century ago to promote the discipline of

microbiology and its role is now more important than ever. The

national leadership is conscious of the need to adapt and change

and has been moving in the past few years to do so, with gathering

momentum. One of our most important platforms is our national

meeting, developed to promote the exchange of ideas. The mem-

bership of the society is increasingly drawn to other conferences to

meet their needs and this must be recognised and accommodated.

Our approach tomeetings including our national scientificmeeting

must evolve with it, as in comparable societies.

We now see increased centralisation and automation and much

greater incorporation of molecular diagnostics and other aspects of

biotechnology in industry, environmental and diagnostic microbi-

ology. Accordingly, a national meeting under the auspices of the

ASM togive aplatform todiscuss research anddevelopment in these

fast-moving areas and to provide workshops for skill development

is being explored, initially focusing on clinical diagnostic microbi-

ology. This follows the development of an additional ASM Travel

award for clinical microbiologists (http://www.theasm.org.au/

awards/asm-clinical-microbiology-travel-award/) and theLynGilbert

Award (http://www.theasm.org.au/awards/asm-lyn-gilbert-award/)

for contributions in clinical microbiology, awarded for the first time

in 2014.

Highly specialised meetings are important and essential for career

development and networking and for exchange of the latest infor-

mation between experts in fast-moving and competitive areas of

endeavour. Deep and narrow in scope by definition, attendance at

these to the exclusion of broader conversations may not meet the

complete needs of early career microbiologists. We must therefore

not only embrace and nurture new directions in microbiology but

be open to ideas coming from outside that which we have long

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 meet-

ings. 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.

A link to state branches and the Visiting Speaker program and other

well-established infrastructure is another easy route that ASM pro-

vides for members to work through new proposals, and give early

career researchers and professional microbiologists a taste of con-

ference organising and a chance to test ideas. This ismanaged easily

through state branches and can be easily progressed to a national

meeting if the idea demands it. The relationships between states

and the utility of the Visiting Speaker Program has been enhanced

by a clearer pathway for VSP engagement, available on the website

(http://www.theasm.org.au/events/visiting-speakers-program/) and

by more formal networking between the state branches, beginning

in 2015.

A key part of our renaissance is a review of governance and

organisational structure, which also begins in 2015, and the realign-

ment of Divisional Chair responsibilities. Nominations for new

Chairs for 2017 are actively sought and will be a key part of the

repositioning of ASM. The revisedConstitution is due to be released

to all members shortly and to be voted on in the July 2015 AGM at

the Canberra meeting.

How can we participate in this rebirth and strengthenmicrobiology

as a discipline in a competitive environment? Readwidely, talk freely

with your colleagues inside and outsidemicrobiology, and promote

the community by supporting ASM: join state branches and national

council, apply formembership, including professionalmembership

andFellowship, developnew ideas formeetingswith your branchor

national office, and honour your colleagues by commending them

for awards and honorary memberships.

The discipline of microbiology is somewhat different to what it was

when theSociety began in 1959 and theSocietymust keeppacewith

it. ASM must better support progressive specialisation of the entire

membership by supporting specialised meetings, some now long-

established and completely autonomous and others that are yet be

conceived. We must at the same time enrich this with wider

engagement and bring into our community those who do not see

themselves as Microbiologists and yet work with us. The role of

the ASM is not to push back against this natural evolution but to

foster it.

Vertical Transmission

2 10.1071/MA15001 MICROBIOLOGY AUSTRALIA * MARCH 2015

Mammalian microbiomes

Linda L Blackall

Email: [email protected]

Endothermic (an organism that maintains its body at a metabolicallyfavourable temperature) amniotes (who lay their eggs on land or retainthe fertilised egg within the mother), also known asmammals, countamong their cohort the largest (whales) and the most intelligent (someprimates, cetaceans and elephants) animals on Earth. However, none ofthe 5488mammalian species live alone since they all support a complexmenagerie of microbes including prokaryotes (Bacteria and Archaea),microbial eukaryotes, and viruses. That so-called ‘microbiome’ playsmyriad roles ranging from the very well known and well studied(disease) through to provocative involvements (mood alteration andbrain activity). Indeed even the microbiome has been subdivided bysome into the bacteriome, the mycobiome and the virome. It is verytimely that this issue be devoted to mammalian microbiomes since thestudy of microbiomes is going through an unprecedented revolutiondue to current and projected capabilities to generate metagenomesequences, determine metatranscriptomic, metaproteomic and meta-metabolomic information and crucially, analyse the deluge of data andinterpret findings ecologically. The novel procedures are broadly in theeconomic realm of numerous researchers, but many do pose consid-erable technical challenges. Thepractical outcomes for host species andtheir environments are diverse.

Fundamental and applied hostmicrobiome research began very early inthe history of microbiology. Indeed, Antonie van Leeuwenhoek(1632–1723), ‘the father of microbiology’, observed and reported onwhat were large selenomonads from the human mouth in 1676. Morerecently, Robert Hungate1 (1906–2004), ‘the father of rumen micro-biology’, developed critical techniques that allowed the study of anaer-obic microbes – the roll tube technique2. Using this method that hemeticulously described, he explored methanogenesis and cellulosebiochemistry (among other metabolic functions) in ruminants and themicrobial ecology of monkey and human guts. The practical outcomesof improved milk, meat and wool production were major drivers torumen microbiology studies.

It has been nearly four decades since ‘Sanger DNA sequencing’ wasintroduced3, the first organism was sequenced4 and ribosomal RNAanalysis was used to determine the third domain of cellular life on Earth,the Archaea5. Since the late 1970s, substantial method developmentsincluding polymerase chain reaction (PCR), improved acquisition ofDNA sequences (automated DNA sequencing) and their analyses haveoccurred. The first decade of this century was part of that methodimprovement and subsequent data eruption. These fundamental

discoveries in the late 1970s were paramount in facilitating mammalianmicrobiome research.

The diversity of prokaryotes in a plethora of environments could becomprehended by applying massively parallel high throughputDNA sequencing methods (starting with 454 Life Sciences‘pyrosequencing’ in 2005) to small subunit rRNA genes. Full genomeswere determined in large numbers (currently of 31,241 prokaryotes),whole-genome phylogenies were reported, and the ‘meta-omics’ fieldsof endeavour were well and truly spawned into the general arena of theMicrobiome. The giddy rate of progress in omics is difficult to keeppace with but critical questions about microbial function and dynamics(stable and mobile) and the chemical interplay between microbes andtheir hosts should be overriding drivers of their investigation, particu-larly in mammals. Pondering the future of microbiome research, acollection of authors recently reported on their individual opinions6,and to quote from this paper:

Overall, future microbiome research regarding the mole-cules and mechanisms mediating interactions betweenmembers of microbial communities and their hosts shouldlead to discovery of excitingnewbiology and transformativetherapeutics.

The articles in thisMicrobiology Australia issue cover a broad range ofmammalian microbiome studies in Australia. The majority are onhumans (oral and gut), but marine mammals (skin, gut and respiratorytract including blowhole), ruminants and terrestrial Australian nativeanimals (oral and gut) are also explored. The motivations for the host-microbe studies in these papers cover the host species fromboth healthand well-being perspectives as well as from general ecological andanimal production improvement viewpoints. Monotreme microbialecology and potential biotechnological discoveries from the consump-tion of toxic diets (e.g. those high in essential oils like Eucalyptus spp.)should attract more attention and are of essential Australian relevance.

References1. Hungate, R.E. (1966) The Rumen and its Microbes. Academic Press, New York.

2. Hungate, R.E. and Macy, J. (1973) The roll-tube method for cultivation of strict

anaerobes. Bulletins of the Ecological Research Committee 123–126.

3. Sanger, F. et al. (1977) DNA sequencing with chain-terminating inhibitors. Proc.

Natl. Acad. Sci. USA 74, 5463–5467. doi:10.1073/pnas.74.12.5463

4. Sanger, F. et al. (1977) Nucleotide sequence of bacteriophage PHICHI174 DNA.

Nature 265, 687–695. doi:10.1038/265687a0

5. Woese, C.R. and Fox, G.E. (1977) Phylogenetic structure of prokaryotic domain –

primary kingdoms. Proc. Natl. Acad. Sci. USA 74, 5088–5090. doi:10.1073/

pnas.74.11.5088

6. Waldor, M.K. et al. (2015) Where next for microbiome research? PLoS Biol.

doi:10.1371/journal.pbio.1002050

BiographyLinda L Blackall is a microbial ecologist who has studied manydifferent complex microbial communities ranging from host associatedthrough to free living in numerous environments. Her research hascoveredmammalian microbiomes spanningmarsupials, humans, rumi-nants and horses and the methods used allow elucidation of massivemicrobial complexity and function in these diverse biomes. She is aProfessor of Biosciences at Swinburne University of Technology in theFaculty of Science, Engineering and Technology.

GuestEditorial

MICROBIOLOGY AUSTRALIA * MARCH 2015 10.1071/MA15002 3

Methane matters in animals and man: frombeginning to end

Emily HoedtA, Paul EvansB, Stuart DenmanC, Chris McSweeneyC, Paraic ÓCuívD and Mark MorrisonD,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 themammalian gastroin-

testinal tract have long been recognised for their capacity to

participate in interspecies hydrogen transfer,with commen-

surate positive effects on plant biomass conversion. Howev-

er, there is also stillmuch to learnabout thesemethanogenic

archaea in regards to their metabolic versatility, host adap-

tation, and immunogenic properties that is of relevance to

host health and nutrition.

Methane, man and best laid plansThe methane club has an exclusive membership, principally

restricted to the Domain Archaea and more specifically, the

Euryarchaeota. Five orders of methanogens have long been recog-

nised: the Methanopyrales, Methanococcales, Methanobacteriales,

Methanomicrobiales, andMethanosarcinales1.However, themember-

ship has recently been expanded to include the Methanocellales2

as well as the provisionally named ‘Methanoplasmatales’3. Members

of the methane club are very popular, invited to join virtually all

anaerobic microbial communities and especially those where

sulphate is limiting. Popular hangouts includemoist soil biomes, fresh

water sediments and rice paddies, landfills, the gastrointestinal tracts

of invertebrate and vertebrate animals, anaerobic lagoons and waste

management facilities4–6. Indeed, the number and distribution of

these hangouts have dramatically increased in recent decades in

response to human population growth and urbanisation, as well as

the intensification of agriculture to feed a hungry world; but the

hangoverhasarrived.Wearenowbeingchallengedtoreducemethane

gas emissions, and in particular, methane emissions from livestock

production systems, which are attributed with producing ~20% of

global methane emissions7, in response to global concerns about our

impacts on the environment and climate change. Additionally, the

resurgent interest in themicrobiota we share our body with, and their

In Focus


impacts on our health and well-being, extends to the methane club8.

For these reasons, there is a renewed interest in gut methanogens,

but is it more of the same or something new? We contend that there

is still much to be learned about members of the methane club

and their behaviour in the digestive tracts of animals and man, from

beginning to end.

Separating the sheep from the goats: methane

and livestockAs herbivores, ruminants rely upon their microbial communities

within the rumen-reticulum to not only deconstruct plant biomass,

but provide the schemes of anaerobic fermentation necessary to

support the formation of protein-yielding and energy-yielding

nutrients such as microbial biomass and short chain fatty acids9,10.

Methanogens have long been recognised to support these process-

es via minimising pH2, with the concept of ‘interspecies hydrogen

transfer’ (IHT) first demonstrated by Bryant and Wolin11 using

culture based experiments with rumen bacteria and methanogens.

Much of the subsequent research focused on the taxonomic and

ecological variations among methanogen communities as affected

by diet, animal breed, and production system. In general terms,

these studies have shown that while autotrophic Methanobrevi-

bacter spp. are often numerically predominant there is also a

relatively diverse population of heterotrophic archaea present in

these animals6,12. In recent years, the application of ‘omics’

approaches has provided new insights into the roles the archaea

might play in rumen function. Poulsen et al. (2013) showed that

the reduced methane emissions from dairy cows fed rapeseed

oil could be attributed to a selective suppression of the

‘Methanoplasmatales’, with coincident decreases in transcripts

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

respect to methane production, and demonstrated that the trait

is heritable14. Using a combination of metagenomic and metatran-

scriptomic methods they found no significant differences in total

methanogen numbers between the ‘low’ and ‘high’ methane

producers, although there were differences in the relative abun-

dances of the methylotrophic Methanosphaera spp. (increased in

‘low methane’ sheep) and the hydrogenotrophic Methanobrevi-

bacter gottschalkii clade (increased in ‘high methane’ sheep). The

metatranscriptomic data revealed that 7/10 genes coordinating

the hydrogenotrophic pathway were significantly increased in high

methane producing sheep. Collectively, these findings suggest

that while the inhibition of select populations of methanogens can

mitigate livestock methane emissions, it is also a heritable trait,

suggesting host-mediated effects on the rumen microbiota. In that

context, ‘high methane’ emitting animals have been postulated

to possess a longer retention of feed within the rumen as well as

alterations in the bacterial ‘ruminotype’ increasing the levels of

ruminal hydrogen, with coordinate elevated expression of

genes encoding the hydrogenotrophic pathway and greater

methane yield15,16. It seems intuitive then to further suggest that

the increased relative abundance of hydrogen-dependent

methylotrophic methanogens in ‘low methane’ animals relates to

their capacity for growthwhen the bacterial ruminotype favours less

hydrogen production during fermentation12,17.

Differences downunder: the low methane

emitting macropodidsThemacropodids (kangaroos, wallabies, pademelons and relatives)

bear some similarity to ruminants in so far as their reliance on

forestomach colonisation bymicrobes for plant biomass conversion

and nutrient provision. In contrast, the foregut microbiota resident

in these animals releases relatively low amounts of methane com-

pared to sheep18,19. Although these observations were initially

proposed to reflect the absence of methanogenic archaea within

the macropodid forestomach, several studies have now demon-

strated the presence of Methanobrevibacter, Methanosphaera,

and ‘Methanoplasmatales’ archaea, albeit at numbers substantially

less than found for ruminant livestock (~106 g.sample–1 c.f. ~108 g.

sample–1)6. Our group has now produced an axenic culture of

Methanosphaera sp. (strain WGK6) from foregut digesta collected

from a Western grey kangaroo (Macropus fuliginosus). Like

the human strain Methanosphaera stadtmanae DSM-3091, WGK6

uses methanol for methane formation, energy production and

growth. However, the annotated draft sequence of the WGK6

genome suggests the macropodid isolate possesses some unique

features that may support a greater metabolic versatility than

previously characterised from studies of the human strain. So

it seems that the adaptations to herbivory in the ‘low methane’

emitting macropodids includes the maintenance of Methano-

sphaera spp., which also seem to be present in greater abundance

in ruminant animals individually confirmed to be ‘low methane’

emitters.

Humans and methanogens: a docile partnership

or secret frenemies?Methanogens are consistently identified from human subjects

deemed healthy or suffering from disease; however the relation-

ships between the diversity of methanogen community members

and the health status of the host are still unclear. Early studies

determined that like other mammals the human large bowel

was colonised by hydrogenotrophic Methanobrevibacter spp.

(principallyMbb. smithii) and themethylotrophicMethanosphaera

spp. (principallyMsp. stadtmanae20). More recently, the analysis of

human microbiota samples from subgingival, intestinal or vaginal

mucosae has further expanded the diversity of methanogenic

archaea to include a new species of Methanobrevibacter (Mbb.

oralis), as well as two isolates of methylotrophic archaea (Candi-

datus ‘Methanomethylophilus alvus’ and Methanomassiliicoccus

luminyensis) affiliated with the newly defined orderMethanoplas-

matales21,22. Interestingly, our own unpublished studies, as well

as the findings of Poulsen et al.13, Dridi et al.21 and Borrel et al.22

show these archaea are capable of using methylated amines arising

from phosphatidylcholine metabolism to support growth. In that

context, establishment of the Methanoplasmatales in the human

large bowel might be of clinical relevance for persons known to

In Focus

MICROBIOLOGY AUSTRALIA * MARCH 2015 5

possess relatively high levels of trimethylamine-oxide in blood,

because of its association with cardiovascular disease pathogenesis

(reviewed by Morrison23 and Brugère24). However, there is also

mounting evidence from cross-sectional studies that variations in

archaeal communities at different body sites might impact human

health25–27. For instance, patients with periodontitis have been

found to harbour large numbers of methanogenic archaea, in

addition to acetogenic and sulphate-reducing bacteria within sub-

gingival periodontal pockets28. Blais Lecours et al.29 also confirmed

that both Mbb. smithii and Msp. stadtmanae can be immunosti-

mulatory in animal models of respiratory disease, with the latter

provoking a stronger immune response. Furthermore,Blais Lecours

et al.30 reported that while the total numbers of methanogenic

archaea are less in patients suffering from inflammatory bowel

disease (IBD), the prevalence of Msp. stadtmanae was greater

in these patients, and healthy human subjects produced an

antigen-specific IgG response to this archaeon. These results

suggest that Msp. stadtmanae prevalence and/or abundance may

be a biomarker of gut dysbiosis, being more prevalent in persons

with an altered ‘low hydrogen’ fermentation scheme. This hypoth-

esis warrants more detailed examination as part of well-designed

clinical studies of IBD and perhaps, other chronic inflammatory

diseases.

SummaryDespite the widespread recognition of the roles methanogenic

archaea play in gut environments, there is still much to learn about

their metabolic versatility, host adaptation, and immunomodula-

tion. Recent research of the methylotrophic archaea from three

divergent mammalian hosts suggests that methane matters in

animals and man, from beginning to end!

References1. Anderson, I. et al. (2009) Genomic characterization of methanomicrobiales

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pone.0005797

2. Sakai, S. et al. (2008) Methanocella paludicola gen. nov., sp. nov., a methane-

producing archaeon, thefirst isolate of the lineage ‘RiceCluster I’, andproposal of

the new archaeal order Methanocellales ord. nov. Int. J. Syst. Evol. Microbiol. 58,

929–936. doi:10.1099/ijs.0.65571-0

3. Paul, K. et al. (2012) ‘Methanoplasmatales,’ Thermoplasmatales-related archaea

in termite guts and other environments, are the seventh order of methanogens.

Appl. Environ. Microbiol. 78, 8245–8253. doi:10.1128/AEM.02193-12

4. Liu, Y. andWhitman,W.B. (2008)Metabolic, phylogenetic, andecological diversity

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annals.1419.019

5. Edwards, T. and McBride, B.C. (1975) New method for the isolation and identi-

fication of methanogenic bacteria. Appl. Microbiol. 29, 540–545.

6. Evans, P.N. et al. (2009) Community composition and density of methanogens

in the foregut of the Tammar wallaby (Macropus eugenii). Appl. Environ.

Microbiol. 75, 2598–2602. doi:10.1128/AEM.02436-08

7. Lowe, D.C. (2006) Global change: a green source of surprise. Nature 439,

148–149. doi:10.1038/439148a

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

9. Karasov, W.H. and Carey, H.V. (2009) Metabolic teamwork between gut microbes

and hosts. Microbe 4, 323–328.

10. Hobson, P.N. (1988) The RumenMicrobial Ecosystem, First edn. Elsevier Applied

Science, New York.

11. Bryant, M.P. andWolin, M.J. (1975) Proceedings of the first international congress

of the international association of the microbiological society, Developmental

Microbiology E, Science Council of Japan, Tokyo, Japan. p. 297.

12. Janssen, P.H. and Kirs, M. (2008) Structure of the archaeal community of the

rumen. Appl. Environ. Microbiol. 74, 3619–3625. doi:10.1128/AEM.02812-07

13. Poulsen, M. et al. (2013) Methylotrophic methanogenic Thermoplasmata impli-

cated in reduced methane emissions from bovine rumen. Nat Commun 4, 1428.

doi:10.1038/ncomms2432

14. Shi, W. et al. (2014) Methane yield phenotypes linked to differential gene

expression in the sheep rumen microbiome. Genome Res. 24, 1517–1525.

doi:10.1101/gr.168245.113

15. Janssen, P.H. (2010) Influence of hydrogen on rumen methane formation and

fermentation balances through microbial growth kinetics and fermentation

thermodynamics. Anim. Feed Sci. Technol. 160, 1–22. doi:10.1016/j.anifeedsci.

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16. Kittelmann, S. et al. (2014) Two different bacterial community types are linked

with the low-methane emission trait in sheep. PLoS ONE 9, e103171. doi:10.1371/

journal.pone.0103171

17. Attwood, G.T. et al. (2011) Exploring rumen methanogen genomes to identify

targets for methane mitigation strategies. Anim. Feed Sci. Technol. 166–167,

65–75. doi:10.1016/j.anifeedsci.2011.04.004

18. Madsen, J. and Bertelsen, M.F. (2012) Methane production by red-necked walla-

bies (Macropus rufogriseus). J. Anim. Sci. 90, 1364–1370. doi:10.2527/jas.2011-

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19. von Engelhardt, W. et al. (1978) Production of methane in two non-ruminant

herbivores Comp. Biochem. Physiol. Part A. Physiol. 60, 309–311. doi:10.1016/

0300-9629(78)90254-2

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.

Microbiol. 141, 116–122. doi:10.1007/BF00423270

21. Dridi, B. et al. (2012) Methanomassiliicoccus luminyensis gen. nov., sp. nov., a

methanogenic archaeon isolated from human faeces. Int. J. Syst. Evol. Microbiol.

62, 1902–1907. doi:10.1099/ijs.0.033712-0

22. Borrel, G. et al. (2012)Genome sequence of ‘CandidatusMethanomethylophilus

alvus’ Mx1201, a methanogenic archaeon from the human gut belonging to a

seventh order of methanogens. J. Bacteriol. 194, 6944–6945. doi:10.1128/

JB.01867-12

23. Morrison,M. (2013) Looking large, tomakemore, out of gutmetagenomics.Curr.

Opin. Microbiol. 16, 630–635. doi:10.1016/j.mib.2013.10.003

24. Brugère, J.F. et al. (2014) Archaebiotics: proposed therapeutic use of archaea to

prevent trimethylaminuria and cardiovascular disease. Gut Microbes 5, 5–10.

doi:10.4161/gmic.26749

25. Furnari, M. et al. (2012) Reassessment of the role ofmethane production between

irritable bowel syndrome and functional constipation. J. Gastrointestin. Liver Dis.

21, 157–163.

26. Pimentel, M. et al. (2003) Methane production during lactulose breath test is

associated with gastrointestinal disease presentation. Dig. Dis. Sci. 48, 86–92.

doi:10.1023/A:1021738515885

27. Lepp, P.W. et al. (2004) Methanogenic Archaea and human periodontal disease.

Proc. Natl. Acad. Sci. USA 101, 6176–6181. doi:10.1073/pnas.0308766101

28. Vianna, M.E. et al. (2008) Quantitative analysis of three hydrogenotrophic micro-

bial groups, methanogenic archaea, sulfate-reducing bacteria, and acetogenic

bacteria, within plaque biofilms associated with human periodontal disease.

J. Bacteriol. 190, 3779–3785. doi:10.1128/JB.01861-07

29. Blais Lecours, P. et al. (2011) Immunogenic properties of archaeal species found

in bioaerosols. PLoS ONE 6, e23326. doi:10.1371/journal.pone.0023326

30. Blais Lecours, P. et al. (2014) Increased prevalence ofMethanosphaera stadtma-

nae in inflammatory bowel diseases. PLoS ONE 9, e87734. doi:10.1371/journal.

pone.0087734

BiographiesEmily Hoedt is a PhD student at The University of Queensland

SchoolofChemistry andMolecularBiosciences, and is supervisedby

In Focus

6 MICROBIOLOGY AUSTRALIA * MARCH 2015

Mark Morrison, Phil Hugenholtz and Gene Tyson. Her PhD studies

focus on functional and comparative studies of heterotrophic

methanogens from different gut environments, supported by an

Australian Postgraduate Award and a top-up scholarship from Meat

and Livestock Australia.

Paul Evans is a Postdoctoral Fellow, based at the Australia Centre

for Ecogenomics at University of Queensland, Brisbane and his

research interests are related to microbial ecology in anaerobic

environments. His research involves examinations of novel

microbes from a range of environments including ruminants, per-

mafrost soils and coal bed methane aquifers. Paul is specifically

interested in archaea that populate these anaerobic environments

and how they interact with other members of the microbial com-

munity to produce energy and make their living.

Stuart Denman is a research scientist with the CSIRO in the

Agriculture Flagship. He has been actively involved in projects that

use molecular methods to detect and monitor key microbial popu-

lations within the rumen. His current research focus is onmicrobial

metagenomics and uses advanced molecular and bioinformatic

techniques to ascertain the interactions and functional changes

that take place in the rumenmicrobiome as they pertain tomethane

abatement strategies.

Chris McSweeney is a Senior Principal Research Scientist at

CSIRO and leads research into the gut microbiology of livestock,

humans and native animals. His current research is focussed on the

molecular basis of hydrogenotropy in gut microbial ecosystems

with emphasis on the function ofmethanogenic archaea. The aim is

to eventually modify the ecosystem to reduce methane emissions

from ruminant livestock.

Dr Páraic ÓCuív is a gut microbiologist at The University of

Queensland Diamantina Institute where he has a long standing

interest in host-microbe interactions as they relate to the aetiology

of chronic gut diseases. He is an expert in gut microbiology,

microbial genetics and functional metagenomics and he currently

leads the isolation and functional characterisation of microorgan-

isms from the human gut as part of the Australian Healthy Micro-

biome Project.

Professor Mark Morrison is trained as a microbiologist with a

specific interest in the role thatmicrobes play in affecting the health

and well-being of humans and animals. After nearly 20 years within

US academia, he returned to Australia in 2006 initially as a Science

Leader within CSIRO, and now as Chair and Group Leader in

Microbial Biology and Metagenomics at The University of Queens-

land Diamantina Institute. His work since returning to Australia

has emphasised the use of ‘omics’ technologies to produce new

insights into the microbial world, and from which, improved meth-

ods for monitoring and adjustment of the gut microbiota might

be achieved; with the goal of improving host animal health and

well-being.

In Focus


The marine mammal microbiome: currentknowledge and future directions

Marine mammals are globally significant because of their

sensitivity to environmental change and threatened status,

often serving as ‘ecosystem sentinels’1. Disease is a major

cause of marinemammal population decline and the role of

the microbiome in disease has generated considerable

interest. Recent research in humans has greatly enhanced

our understanding of how the host-associated microbial

community, the microbiome, affects host health. In this

review, we provide an overview of the extent of the marine

mammal microbiome with a focus on whole community

characterisation using genomic methods. This research

highlights the overlap in microbial communities between

geographically distinct species and populations of marine

mammals, suggesting tight links betweenmarinemammals

and their microbial symbionts over millions of years of

evolution. An understanding of these links in both healthy

and compromised hosts is essential to identifying at-risk

populations and making ecologically appropriate manage-

ment decisions. We advocate further development of

innovative sampling and analytic techniques that advance

the field of microbial ecology of marine mammals.

Recent investigations have highlighted the capacity of the micro-

biome to act strongly and significantly in maintaining host health

with a vital role in disease manifestation and immune system

function2,3. Members of the microbial community can directly

influence the progression of a disease via infection and also mod-

ulate the host’s own immune system regulation and response4.

Indeed thehost’smicrobial partners areessential to immunesystem

function. Themicrobiome has been observed to be species-specific

in a variety of vertebrate hosts5–7 and is influenced by host phylog-

eny, as a result ofmillionsof yearsof co-evolution8.Marinemammals

represent unique evolutionary lineages and investigations into their

associated microbes will provide a deeper understanding of their

ecology and evolution.

Marine mammals form a diverse group of 129 species in three

orders, and of those, 28 are considered endangered or threatened9.

Disease is one of the main causes of death in marine mammals and

somepopulationshave sufferedmassmortalities causedbybacterial

pathogens10. Bacteria exist as part of the normal, or even beneficial,

flora associated with a host, fluctuating and changing with a host’s

physiology andmetabolism11. Inmammals, disease canoccur under

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]

In Focus


a number of different circumstances, most commonly on occasions

when the host’s immune system is compromised. For marine

mammals, susceptibility to pathogens may be particularly elevated

due to anthropogenic stressors such as depleted food resources,

habitat degradation and chemical or sound exposure12–15. Addi-

tionally, succession events occurring after an initial bacterial infec-

tionmay lead to dysbiosis, and alterations in the host’s microbiome

may be a better predictor of disease progression than following the

presence of individual pathogenic agents16. Hence, we need to

establish baseline data on microorganisms commonly associated

with marine mammals in order to detect anomalies. In the last

decade genomic sequencing technologies have provided a previ-

ously unrecognised diversity of microorganisms in numerous

diverse habitats. In this brief review we highlight the current

knowledgeof themicrobial composition in associationswithmarine

mammals with a focus on whole community characterisation.

Skin microbiomeSkin, 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-

ders, however the role of microorganisms in these conditions is

still largely unresolved and knowledge is primarily founded on

cultivation-based studies17. The recent application of cultivation-

independent sequencing-survey approaches to humpback whale

(Megaptera novaeangliae) skin has demonstrated that a unique

ecosystem of microbes resides on the skin surface (Table 1), which

differs from the community present in seawater18.

Among populations of humpback whales surveyed in diverse geo-

graphic regions, two genera of bacteria (Bacteroidetes genus Tena-

cibaculum and Gammaproteobacteria genus Psychrobacter) were

found to be cosmopolitan and abundant associates on humpback

whale skin26. Scanning electron microscopy of humpback whale

skin revealed a rich layer of microbial cells on the skin surface26, but

ashumpbackwhales regularly undergo skin sloughing throughboth

behavioural27 and physiological activities28 it is possible that the

robust Tenacibaculum and Psychrobacter cells may have some

means to maintain their residence on the whale skin and could

provide benefits to their host. Sequencing survey-based data also

demonstrate differences between the skin bacterial associates of

healthy and health-compromised humpbacks18,26. Additional data

on and study of the skinmicrobiomemight potentially improve our

ability to assess health status among free-rangingmarine mammals,

in particular cetaceans.

Gut microbiomeThe gastrointestinal tract is home to an abundant community of

microorganisms. The gutmicrobiomeplays a significant role in food

breakdown and digestion, the production of essential vitamins and

minerals and regulationof the immunesystem3. In youngmammals, Table

1.Relativeabundanceofbacterialp

hyla

comparedbetw

eenknownstudiesofmarinemammalspeciesandanatomicalsitesin

healthyindividuals

Order

Cetacea

Carnivora

Sirenia

Sub-O

rder

Mysticeti

Odontoceti

Phocidae

Pinnipedia

Com

mon

name

Hum

pbackwhale

Bottlenose

dolphin

Leopard

seal

Sothern

elephant

seal

Hooded

seal

Harbour

seal

Grey

seal

Australianfurseal

Australian

sealion

Dugong

Manatee

Species

Megaptera

noveangliae

Turisops

truncatus,

T.aduncus,

hybridA

T.truncatus

Hydrurga

leptonyx

Miroungaleonina

Cystophora

cristata

Phoca

vitulina

Halichoerus

grypus

Arctocephalus

pusillusdoriferus

Neophoca

cinera

Dugong

dugong

Trichechus

manatus

latirostris

Age

group

Adult

Calf

Adultand

Sub-adult

NR

Adult

Adultand

sub-adult

Pup

NR

NR

NR

9m

pup

6m

pup

2m

pup

NR

Adult

Adult

Sub-adult

Calf

Sam

ple

Skin

Skin

Blow

Blow

Faeces

Faeces

Faeces

Colon

Colon

Colon

Faeces

Faeces

Faeces

Faeces

Faeces

Faeces

Faeces

Faeces

Bacterial

phyla(%

of

community)

Firm

icutes

1<1

15

4443

1822

5076

8387

8380

8379

7971

Bacteroidetes

4063

134

821

1468

4924

106

42

1517

1926

Proteobacteria

6036

5060

3115

59

00

2<1

48

<1<1

<1<1

Fusobacteria

<1<1

1<1

1320

621

<1<1

<1<1

<1<1

0<1

<1<1

Num

berofindividuals

516

244

1218

69

11

44

41

118

117

Methodology

PP

PCL

PP

PCL

CL

CL

PP

PM

CL

PP

P

Reference

19

19

19

20

77

721

21

21

22

22

22

23

24

25

25

25

Datasummarised

forthedominantbacterialphylaacross

speciesandanatom

icalsites.Tabledataareas

follows:notrecorded(NR);month(m);clonelibraries

(CL);pyrosequencing(P);metagenom

icsequencing

(M).

AHybridbottlenose

dolphinreferstoindividualssiredby

T.truncatustoT.aduncusfemales

born

incaptivity.

In Focus


the gutmicrobiome is required for full development of the immune

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 du-

gong). 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 com-

position of the gut microbiome7,25.

Amongst the seals, the gut microbiome of pinnipeds has a greater

abundance of the phylum Firmicutes compared with phocids

(Table 1). A ‘core’ group of microorganisms including the genera

Ilyobacter, Psychrilyobacter, Fusobacterium, Bacteroides, Subdo-

lingranulum, Sporobacter, Sutterella,Weisella, Anaerococcus and

Campylobacterhavebeenobservedwithinphocid seals7,21,22whilst

their herbivorous relatives, within the order Sirenia, shared mem-

bers from the order Clostridiales, including the genera Clostridium

and Ruminococcus24,25,31. The presence of shared bacterial oper-

ational taxonomic units (OTUs) in multiple hosts from different

studies highlights the strong phylogenetic influence on microbial

assembly.

Respiratory microbiomeRespiratory illnesses such as pneumonia are a major cause of

mortality in both wild and captivemarinemammals32. The cetacean

upper respiratory tract terminates in a blowhole, positioned at the

top of the head. This feature is a unique adaptation to life in the

marine environment, and allows airways to be effectively sealed off

from seawater. Upon surfacing, cetaceans forcefully exhale and in

the process eject a substance termed blow (also called condensed

respiratory vapor or exhaled breath condensate). This material has

been shown toharbour potential pathogens inwhales33 andhas also

been used to characterise the normal respiratory-associated micro-

biome residing in the upper respiratory tract of bottlenose dol-

phins19,20 (see collection methods in Figure 1). Members of the

bacterial genera Plesiomonas, Aeromonas, Escherichia, Clostridi-

um and Pseudomonas, Burkholdaria, Mycobacterium, Haemo-

phylis, Streptococcus and Staphylococcus (including multiple

resistant Staphylococcus aureus) have been detected in both

sick/dead34 and healthy, free-ranging cetaceans20,33,35.

Blow samples from both free-ranging Tursiops truncatus and

captive T. aduncus and T. truncatus were dominated by three

novel dolphin associated clades (termed DAC 1, 2 and 3) within

the Cardiobacteraceae lineage of the Gammaproteobacteria19,20.

The Cardiobacteraceae are facultative anaerobic, Gram-negative

rod-shaped cells, members of which form part of the commensal

microbiome of humans, and whose growth is enhanced by the

presence of carbon dioxide36, which occurs in high abundances at

the termination of the respiratory tract. Representatives from each

of DAC 1, 2, and 3 have been present in every bottlenose dolphin

surveyed thus far, although themajority of sequences are associated

with DAC 3, indicating this is likely a ubiquitous and critical com-

ponent of the dolphin respiratory system. Other ‘core’ taxa asso-

ciated with the dolphin respiratory microbial community appear

to include the Arcobacter, Hydrogenimonaceae, Halotalea, Aqui-

marina, Helococcus, Mycetocola, Methylococcus and Marinimi-

crobium19. Temporal analysis of captive dolphins suggests

community composition in healthy animals is quite stable

and that individual dolphins harbour consistently unique microbial

communities19.

Sampling techniquesSampling of material for microbiological analysis from marine

mammals is logistically challenging (reviewed by Hunt et al.37),

hence themajority of information onmicrobial disease comes from

captiveor strandedanimals that arenotnecessarily representativeof

(a)

(b)

Figure 1. Exhaled ‘blow’ samples provide access to respiratorymicrobiome, host DNA, hormones and associated metabolites.Bottlenose dolphins can be trained to exhale on demand allowingcollections to be made routinely as shown here by Jillian Wisse fromthe National Aquarium in Baltimore, Maryland, USA in captive dolphins(a) and Dr Ewa Krzyszczyk, collecting samples from wild bottlenosedolphins that visit a beach in Shark Bay, WA, Australia (b). Photo creditmonkeymiadolphins.org.

In Focus


the greater wild population. However, current sampling methods

(see examples in Figures 1 and 2) still provide considerable insight

into the microbiome of marine mammals. Capture by sedation or

restraint has been employed on smaller species such as seals and

dolphins7,38,39 and has recently been used for some larger whales40.

However, there are few opportunities to sample using these meth-

ods. It is increasingly common to use biopsy darts for collection of

skin and blubber samples for genetic and, now, microbiological

studies18,41. Permissions for biopsy sampling can be challenging for

some species ofmarinemammals, and repeated samplings areoften

not possible for the same individuals. In order to increase existing

data on the marine mammal microbiome, logistically feasible, non-

or minimally-invasive sampling protocols that are easily reproduc-

ible and provide biological material suitable for a range of studies

are necessary. For example, respiratory blow can be used to

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

the gut microbiome22,23.

Future researchIt appears likely that there are deep branching clades of bacteria

that are uniquely associated with marine mammals and have been

conserved throughout the evolution of their hosts. Many bacterial

sequences obtained from marine mammal studies have close rela-

tives that originate from other marine mammal species. This has

significant implications for the transmission of disease amongst

these hosts. As they are usually highly social animals, there are

numerous opportunities for the transfer of microorganisms

between individuals47. Diseases inmarinemammals have also been

shown tohave their roots inothermammals, includingdogs48,49 and

humans50. In many cases where disease has caused significant

mortality in wild marine mammals, it has been linked to viruses,

includingmorbillivirus, phocine distemper and influenza virus51–55.

Despite these links being made there is really very little known

regarding the ecological role of viruses in marine mammal hosts.

Further investigations into the factors responsible for shaping the

marine mammal microbiome need to be made. Designing studies

that control for host variation will allow us to make headway in our

understanding of disease manifestation. Studies that focus on the

functionality of themicrobiomewill reveal the interactions between

host and the microbial community23,56. In human subjects, similar

target investigations have allowed for the development of novel

metabolites to treat and prevent disease57. Unlike humans, howev-

er, to access adequatebiologicalmaterial, stridesneed tobe taken to

develop innovative andnon-invasive techniques for the collectionof

relevant samples from wild populations.

AcknowledgementsWe thank Dr Ewa Krzyszczyk and Jillian Wisse for allowing us to use

their photographs.

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BiographiesTiffanie Nelson is a researcher from Australia currently undertak-

ing a postdoctoral fellowship atMontana StateUniversity, Bozeman,

USA. Tiff is a microbial ecologist, who focuses on the microbiome

In Focus


of marine mammals as well as humans and environmental

samples. Her interests are in health and disease associated with

the microbiome. Tiff’s current project is investigating the vaginal

tract microbiome of women in relation to bacterial vaginosis using

both culture-dependant and -independent methods.

Amy Apprill is a researcher at the Woods Hole Oceanographic

Institution in Massachusetts, USA. Amy is a marine microbiologist

researching questions that focus on the contributionofmicroorgan-

isms to the health and ecology of marine animals. Amy is also

interested in how animal-associatedmicrobes reflect the alterations

occurring in their surrounding marine environment. Her current

research uses a combination of field measurements and observa-

tions and laboratory experiments and reliesondiversemethodology

(cultivation, genomic, metagenomic and bioinformatic) to examine

the microbiomes of reef-building corals and marine mammals.

Janet Mann is professor of biology and psychology and vice

provost for research at Georgetown University, Washington DC,

USA. Janet has expertise in the field of animal behavior with

extensive research focusing on marine mammals. Her work has

focusedon social networks, female reproduction, calf development,

life history, conservation, tool-use, social learning and culture

among bottlenose dolphins in Shark Bay, Australia. Her long-term

study ‘The Shark Bay Dolphin Research Project’, tracks over 1600

dolphins throughout their lives and includes an international team

on three continents where each group studies different aspects of

delphinid biology.

Tracey Rogers is associate professor at the University of New

South Wales. Tracey works across a diverse range of research fields

with many years of experience working in Antarctica with marine

mammals. The common theme inTracey’s research is in attempting

to understand how mammals respond to change. Tracey uses

multidisciplinary approaches to understand the ecology of mam-

mals. Most of her work uses models and techniques with captive

populations for applications in field settings. Other techniques

include stable isotope analysis, satellite telemetry and acoustics.

Mark Brown is a senior research fellow at the University of

New South Wales, Sydney, Australia. He has extensive expertise in

research that focuses onmicrobes (Bacteria, Archaea andmicrobial

Eukaryotes), primarily from marine environments. Mark’s main

interest is in investigating how microbes interact with each other

and their environment to form communities that sustain critical

ecosystem processes. His current research couples innovative

in situ sampling methods, genetic tools, bioinformatics and eco-

logical theory to elucidate and predict the form, function and

impact of microbes in rapidly changing ecosystems.

In Focus


The role of the gut microbiome in host systems

Clarissa FebiniaA, Connie HaA, Chau LeA and Andrew HolmesA,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

on animals that they are best considered holobionts – an

organism comprised of multiple biological partners. The

concept of dysbiosis is disease states that result from unde-

sirable interactions between the partners in a holobiont.

Many molecular mechanisms that link the gut microbiome

with host health and disease have now been established and

these are giving rise to new insights in healthcare. In essence

these studies show that our microbiome is so closely inter-

twinedwith our physiology thatmicrobiome composition is

reflective of many aspects of our health. Of special impor-

tance is recognition of the intersection between chronic diet

habits and the microbiome in driving changes in our phys-

iological state. In the foreseeable future it is likely micro-

biome profiling will be a standard diagnostic test in diverse

areas of medicine and that interventions targeting the

microbiome will be developed.

All animals are associated with microorganisms for the majority of

their life, only embryonic stages are microbe-free. However the

complexity of animal-microbe interactions and the nature of their

outcomes vary. For some animals their associations with microbes

include obligate partnerships with a specific microbe that has

obvious benefits for the animals life history (e.g. Coral:Zooxanthel-

lae, Squid:Vibrio, Aphid:Buchnera). For others, the animal may

have a specialised structure in which it receives obvious benefits

frommicrobes (e.g. the rumen), but these arise via a community of

many microbial species. In contrast microbes can also interact with

animals to cause disease. For the majority of animals such specific

pathogens have historically been the focus of scientific attention.

The remaining microbes were traditionally viewed as commensals.

The past decade has seen a dramatic, and ongoing, revision of this

view with recognition that those microbes that form communities

of stable composition at various body sites (our microbiomes)

influence many aspects of our postnatal development and physiol-

ogy. This is especially true of the gut microbiome.

The links between the gut microbiome and host physiological

properties are now known to be important to the pathophysiology

ofmetabolic and immunological diseases. Studies of germ-free (GF)

animals have demonstrated robust connections between the gut

microbiome and host development and physiology. These include

roles in vascular development1 and immune cell maturation2,3.

A consequence of such developmental effects is that emergent

aspects of animal health andphysiology such as inflammatory tone4,

energy balance5,6, feeding behaviour and even mood and gross

anatomy can differ in germ-free animals. Three key points that have

emerged from these studies are schematically represented as they

might apply to human biology in Figure 1. First, the existence of GF

animals indicates the presence of microbes is not essential for the

viable development and physiology of an animal. However, GF

animals are different, with significant constraints on their environ-

mental fitness, including susceptibility to systemic infection should

they be exposed to pathogens and having additional nutritional

demands (Figure 1a). Second, if microbes are non-essential to

normal physiological processes of animals it is arguable that their

most fundamental contribution to the animals state is alteration of

how the animal system perceives and responds to its environment,

both internal and external. Finally, both microbiome association

studies and transplant studies show that different compositions of

themicrobiome are associated with different host states. Where the

microbiome composition gives benefits to desirable host functions

In Focus


such as improved nutrition on available foods or reduced immu-

nopathology we may view the stable host-microbiome system as a

healthy holobiont (Figure 1b1). Where the microbiome composi-

tion is stable, but results in undesirable host functions such as

impaired energy balance or inflammation we may view the holo-

biont as being in a state of dysbiosis (Figure 1b2).

Although various studies have proposed some key beneficial

microbes for gut health (e.g. Faecalibacteriumprausnitzii,Copro-

coccus sp., Ruminococcus bromii, Bacteroidetes spp.), it is rare

that presence or absence of any one microbe is specifically

associated with health. This reflects a high degree of functional

redundancy in the gut community, wherebymultiplemicrobes with

similar functions comprise ‘guilds’ that have broadly similar eco-

logical roleswithin the community. Thus either benefit or detriment

to the host system is typically an emergent property of the whole

microbial community. Details of the mechanisms by which

variations in the gut community impact host nutrition and physiol-

ogy are now emerging. Broadly speaking microbes contribute to

nutrition through the production of metabolites and impact phys-

iology through both metabolites and structural components.

Microbial conversion of digestion resistant carbohydrate to short

(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 forhost-microbiome interaction is the intestinal interface where nutrients are absorbed and critical signals for regulation of homeostasis of the animalsystem originate. Animal studies have shown that microbes contribute directly to differences between monobionts (a) and holobionts (b) in thestructure of the intestinal interface and in the breakdown of food. Differences in microbial composition can drive differences in animal health viaimmune and neuroendocrine signalling.

In Focus


chain fatty acids (SCFAs) or production of vitamins both result in

increased capacity for the host system to extract nutrition from

food6,7. The SCFAs also exert other effects in the host system,

particularly butyrate which is a primary energy source for colono-

cytes, and therefore important for maintaining epithelial health.

SCFAs also impact the function of other tissues and organs in

the host by acting as signalling molecules for G-coupled protein

receptors (e.g. GPR41, GPR43). Known regulatory roles of SCFAs

include: appetite regulation, epigenetic state, gut motility, energy

metabolism, endocrine functions, and immune regulation8-10. Since

these SCFAs are primarily microbial metabolites it could be argued

that the host is monitoring the activity of its microbiome via

metabolite sensors and integrating this information into homeo-

static regulation. Similarly thehost alsomonitorsmicrobial presence

viapattern recognition receptors andsignallingpathways contribute

to regulation of diverse aspects of immune andmetabolic functions.

Significantly, disruption of the key metabolite receptors (e.g.

GPR4111) or PRR receptors (e.g. TLR512) inmouseknockoutmodels

is capable of eliciting disease states, highlighting the importance

of microbial signalling for dysbiosis. Collectively these observations

show that change in the nature or strength of various microbiome

signals, rather than presence/absence of specific microbes, is the

primary determinant of health or dysbiosis. Given this, in order

to understand dysbioses we must ask what drives disturbance

to this?

Inwild type animals this signalling-based disturbance to host-micro-

biome interactions is thought to primarily arise through changes in

microbial composition or activity. Since different microbes (e.g.

Gram-positive vs Gram-negative) contain different microbe-associ-

atedmolecular patterns (MAMPs) theydrivedifferent PRR-signalling

pathways. Similarly since microbes differ in their capacities to

degrade macromolecules and which metabolites they produce,

changes in community composition will also drive changes in

metabolite-signalling pathways. Although diverse factors including

anatomical, genotypic, cultural and environmental factors can in-

fluence the gutmicrobial community13–15, it is chronic diet patterns

that are thought to be the dominant factor, since what we eat, and

the pattern of food consumption, impact the availability of nutrients

to gut bacteria for their growth and metabolism. Arguably, the key

insight is not the role of themicrobes, but rather the role of diet as a

key modulator of the interaction between microbes and the

host6,14,16. This reflects that major mechanisms of microbial influ-

ence are via small molecules that are uniquely microbial cellular

components ormetabolites. In summary, the concepts of dysbiosis,

and animals as holobionts, are changing the way we view human

biology, especially modern diseases with a lifestyle component.

In general terms there are two routes to improve health via under-

standing of the microbiome; diagnostics and interventions. In

diagnostics, microbiome signals are included in our evaluation of

the host state to inform disease prognosis or intervention

plans. In intervention the microbiome is itself the target of manip-

ulation (e.g. prebiotics or probiotics). Greater understanding of

host-microbiome interactions can inform both routes through:

(1) identification of biomarkers of health or disease in microbiome

association studies (e.g. cancer diagnostics17); (2) identification

of specific microbes or consortia of microbes that are capable of

effecting change if introduced18–20; or (3) intervention in signalling

pathways that derive from microbes21. Progress toward these

objectives could be achieved across a very wide range of diseases

and conditions if microbial community profiling were broadly

adopted as a standard test. However, standardised experimental

protocols and metadata collection (e.g. sample collection, DNA

extraction method22, diet formula) need to be implemented in

order to discern patterns that are robust across geographically and

culturally diverse populations23.

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In Focus


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BiographiesThe authors are all members of the School of Molecular Bioscience

and the Microbiome node of the Charles Perkins Centre at the

University of Sydney. Their research is focussed on understanding

the dynamics of gut microbial community composition, the

mechanisms of host-microbe interaction in the gut and develop-

ment of tools to enablemanagement of the gutmicrobial ecosystem

forhealth.Aparticular focus is the relationshipbetweenournutrient

environment and its effect on host-microbiome interactions in

health. We gratefully acknowledge funding support from the ARC

and NHMRC.

Clarissa Febinia is a postgraduate student and the recipient of an

Australia Awards Scholarship with affiliations to the Eijkman Insti-

tute forMolecular Biology, Jakarta. Her project is on the intersection

between cultural, genetic and diet factors in lifestyle disease.

Connie Ha is in the final year of PhD studies on themechanisms of

diet-induced obesity and the recipient of an APRA.

Chau Le is a PhD student working on the influence of gutmicrobes

on regulation of feeding behaviour. She is the recipient of an APRA.

Andrew Holmes is currently an Associate Professor in the School

of Molecular Bioscience at the University of Sydney and leads

research programs in the Charles Perkins Centre and Marie Bashir

Institute. He was the recipient of the 2006 Fenner Prize from the

Australian Society for Microbiology. He has general interests in

microbial diversity, its evolutionary origins and ecological applica-

tions. He is a Senior Editor forMicrobiology and The ISME Journal,

and a member of the Editorial Boards of Applied and Environmen-

tal Microbiology and Environmental Microbiology.

In Focus


Modulation of the rumen microbiome

Rosalind GilbertA,B,D, Diane OuwerkerkA,B,E and Athol KlieveB,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

fore-stomach of ruminants (rumen) enable domesticated

ruminants to be sustained on forages and fibrous feedstuffs

that would be otherwise indigestible. Ruminants can also

utilise more easily digestible, high energy plant material

such as grain, to achieve rapid increases in weight gain,

muscle bulk and in the case of dairy cows, high milk yields.

Since the mid-1900s there has been a steady research effort

into understanding the digestive processes of ruminants,

striving to maintain animal health and nutrition whilst

maximising theproductivity andenvironmental sustainabil-

ity of livestock production systems. This article describes

strategies developed to modulate the rumen microbial eco-

system, enabling the utilisation of plant feedstuffs that may

otherwise be toxic and enhancing feed utilisation efficiency

or controlling populations of specific rumenmicrobes, such

as those contributing to lactic acidosis and enteric methane

emissions. It also traces advances in technologies that have

enabledus tounderstand theunderlyingbiologicalmechan-

isms involved in the modulation of the rumen microbiome.

The rumen microbial communityThe rumen contains a dense microbial community that actively

degrades plant material, providing the animal with energy via the

end-products of fermentation (short chain fatty acids) and protein in

the form of microbial protein, which flows from the rumen into the

lower intestine1. Rumen microbes not only adhere to and degrade

plant material they may also utilise substrates produced by other

microbes. The rumenmicrobial population includes bacteria that are

predominantly strict anaerobes with the capacity to be highly fibro-

lytic and proteolytic (generally of the phyla Bacteroidetes, Firmicutes

andProteobacteria),methanogenic archaea (phylumEuryarchaeota)

and anaerobic fungi (fungal division Neocallimastigomycota)2,3.

These rumenmicrobes are predated on by populations of anaerobic

protozoa (predominantly of the phylum Ciliophora)4,5 and viruses

(predominantly dsDNA bacteriophages of the order Caudovirales)6.

Rumen microbes friend or foe? Strategies

for reducing plant toxicity, acidosis

and enteric methaneThe modulation of rumen microbial populations has traditionally

focussedon strategies to improve feeddigestibility and consequent-

ly increase overall animal productivity, reducing the time taken for

ruminant livestock to reach market-weight specifications. Micro-

biologists and animal nutritionists have sought to determine the

impact of different diet formulations on rumen function and live-

weight gains, investigating the effects of feedstuff pre-treatment

employing either physical change (for example steam treatment,

rolling or flaking of grain7–9) or physical and chemical changes

through microbial and enzymatic pre-treatment (for example ensi-

lage of fodder crops with or without the application of silage

inoculants10–12). Research has also sought to increase the environ-

mental sustainability of livestock production by investigating the

Under theMicroscrope


viability of alternative feedstuffs such as those that may also be a by-

product of food production or industry (for example cotton seed

meal13) or feeds that may be readily propagated on-farm (for

example microalgae14,15). Strategies have also been developed to

allow cattle to utilise plant feedstuffs that may otherwise be toxic to

the animal, for example the leguminous shrub Leucaena leucoce-

phala may be propagated on-farm as a high-protein fodder crop

(Figure 1). Leucaena however, contains the toxic amino acid

mimosine and normal rumen microbial degradation results in the

formation of the toxin 3-hydroxy-4(1H)-pyridone (DHP). The ac-

cumulation of these toxins leads to negative health implications

includinghair loss, reduced live-weight gain andgoitre. The solution

to preventing the development of Leucaena toxicity arose from

rumen microbes, as these toxins were first shown to be de-toxified

bybacteria of the genera Synergistes isolated from the rumenof feral

goats16. A mixed microbial drench containing Synergistes jonesii is

currentlyproduced inQueensland for the treatmentof cattlegrazing

Leucaena17.

In addition to strategies to improve feed breakdown in the rumen,

research has also been undertaken to specifically target and control

certain rumenmicrobial populations including the rumenprotozoa,

amylolytic bacteria, and more recently, the methanogenic archaea.

Rumen protozoa may positively contribute to ruminant feed break-

down18; however, as these eukaryotes actively graze on the rumen

bacterial populations, their growth and proliferation within the

rumenmay also contribute to the inefficient, intra-ruminal recycling

of microbial protein5. Strategies to reduce rumen protozoa have

included the use of diet (high grain diets tend to reduce protozoal

populations), dietary additives such as the clay bentonite, nitrates

and vaccination19,20. While these strategies have been shown to

impact onprotozoal populations, it hasprovendifficult to complete-

ly remove protozoa from the rumen.

Strategies have also been developed to control rumen populations

of the amylolytic bacterium Streptococcus bovis. Amylolytic, lactic

acid-producing bacteria such as S. bovis, may over-proliferate in the

rumen when cattle are fed high concentrate or high grain diets,

contributing to the development of a condition known as lactic

acidosis. S. bovis has been targeted through the application of

antibiotics such as monensin21 and phage therapies22. The strate-

gies developed to control S. boviswere largely undertaken between

the 1970s and1990s and the relative importanceof controlling these

organisms and their overall contribution to the development of

lactic acidosis has been cause for debate23,24. Feeding practices

avoiding sudden dietary changes to large quantities of fermentable

carbohydrates can prevent the development of acidosis25 and most

of the novel strategies to prevent rumen acidosis reported in the

literature26,27 havenot beeneither commercialised or implemented

within the livestock production industry.

In the past decade, investigations to modulate rumen microbial

populations have focused on strategies to reduce the amount of the

potent greenhouse gasmethanegeneratedby livestockproduction.

Normal rumen microbial fermentation results in the accumulation

of hydrogen. This hydrogen may be utilised by acetogenic bacteria

(for example of the genera Acetitomaculum, Eubacterium

and Blautia), however the majority of hydrogen is consumed by

populations of methanogenic archaea belonging to the genera

Methanobrevibacter,Methanobacterium,Methanococcus,Metha-

nomicrobium andMethanosaeta, with the methane produced lost

to the animal via eructation28. Strategies currently in development

to control rumen methanogen populations include specific diets

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



(high grain and/or oil), dietary additives (for example naturally-

occurring plant-derived compounds and synthetic anti-methano-

genic compounds), animal breeding, phage-based therapies and

vaccination29–32.While several of these novel approaches have been

shown to be effective in reducing rumenmethanogen populations,

the majority are not yet at the stage of commercial application and

adoption by the livestock production industry.

Feed supplements, probiotics and direct fed

microbialsThe Australian agricultural feed industry produces many feed sup-

plements designed and marketed to improve ruminant production

efficiency, particularly for the dairy industry. Australian law requires

that all agricultural and veterinary chemical products sold in

Australia be registered by the Australian Pesticides and Veterinary

MedicinesAuthority (APVMA,http://apvma.gov.au) andare listedon

the permits and PubCRIS database (https://portal.apvma.gov.au/

pubcris). These feed supplements include direct fed microbials

(DFM) or probiotics and may also incorporate additional enzymes

(amylases, proteases), minerals and salts (selenium, potassium).

There are approximately 30 formulations of probiotic microbes

available for use within Australia to enhance the overall digestive

efficiency of ruminants incorporating bacteria such as Bifidobacter-

ium (including the species bifidum, longum and thermophilum),

Lactobacillus (speciesacidophilus,delbrueckii subspecies bulgar-

icus, plantarum and rhamnosus) and Enterococcus faecium.

In addition, a further 11 registered products are available that are

exclusively basedon the yeast Saccharomyces cerevisiae.While the

survival and proliferation of these organisms in the rumen and the

effect of these probiotic bacteria on rumen digestive processes

has been largely under-represented in the scientific literature, the

probiotic effects of yeasts (S. cerevisiae) has beenmore extensively

assessed33,34. Results can be highly variable between studies, how-

ever several investigations have established that the provision of

these commercially available probiotics ismost useful when applied

to young ruminants to accelerate the establishment of a healthy

gastrointestinal microflora35,36. Probiotics may be used to exclude

undesirable zoonotic pathogens suchasEscherichia coliO157 from

establishing in the ruminant gastrointestinal tract and may also

impact on the ruminant host immune system and feed breakdown

efficiency36–38.

While bacterial strains of rumen origin would be anticipated to

survive and proliferate in the rumen and therefore have a selective

advantage overmicrobes of non-rumen origin36, there are currently

no commercial formulations of rumen-derived probiotic bacteria

registered for use in Australia. The mixed microbial drench for

Leucaena toxicity is the only APVMA approved rumen-derived

microbial treatment. There have however been several reports in

the scientific literature of rumen-derived bacterial isolates being

examined for application as potential probiotics, for example,

Megasphaera eldenii, Ruminococcus sp., R. flavefaciens, Prevo-

tella bryantii, Butyrivibrio fibrisolvens37,39. These strains were

selected for further testing in order to increase starch utilisation

(by the genera Ruminococcus, Prevotella and Butyrivibrio) and to

assist with the prevention of lactic acid accumulation contributing

to acidosis (M. elsdenii) in feedlot cattle.

Future directionsThe success of any strategies developed to modulate the microbial

population of the rumen will always need to be underpinned by

fundamental research efforts to understand how the strategies

impact on the baseline or ‘normal’ functioning of the rumen

microbial ecosystem40. Addressing the gaps in current understand-

ing of the rumen microbial ecosystem41 is therefore key to the

developmentof newstrategies to control themicrobial populations.

The rumen contains a very dense microbial ecosystem, end-

products of microbial digestion, salts and plant material including

partially digested fibre, carbohydrate, and phenolic compounds

and as such, samples of rumen material often present unique

technical challenges. Rumen microbiologists have therefore always

been quick to adopt new developments in technology in order to

more fully understand the complex microbial ecosystem of the

rumen. Early research efforts relied on microbial cultivation and

although the study of cultivated rumen microbes is important for

characterising microbial genera and elucidating their specific

genetic and metabolic traits, culture-independent studies for the

detection of specific microbes (real-time PCR assays) and commu-

nity analysis based on the comparative phylogeny of the prokaryote

16S rRNA gene are often employed to ascertain the extent to which

probiotic microbes survive and proliferate within the rumen31.

Rapid advances in high-throughput sequencing technologies have

also facilitated investigations into how probiotics can influence

both the community composition and functional gene capacity of

the rumen. Metagenomic studies of the rumen have progressed

current understandingof the functional genepotential of the rumen

microbial populationenabling the in silico identificationofenzymes

involved in feed breakdown42.

In the future, a greater reliance on gene sequence-based technol-

ogies or ‘omics’ will lead to an increased understanding of the

interactions occurring between probiotics and the microbial popu-

lations indigenous to the ruminant gastrointestinal tract. This is of

particular interest for the development and optimisation of new

and more effective strategies for the modulation of the rumen

microbiome. Development of new strategies, treatments and pro-

biotics to enhance rumen feed utilisation efficiency, represents an

area of great potential for the Australian livestock industries andwill

enable the production of quality products to meet global demands.

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3. Orpin, C.G. (1975) Studies on the rumen flagellate Neocallimastix frontalis.

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7. Owens, F.N. et al. (1997) The effect of grain source and grain processing on

performance of feedlot cattle: A review. J. Anim. Sci. 75, 868–879.

8. Yang,W.Z. et al. (2001) Effects of grainprocessing, forage to concentrate ratio, and

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9. Yang, W.Z. et al. (2013) Quality and precision processing of barley grain affected

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26. Klieve, A.V. et al. (2012) Persistence of orally administeredMegasphaera elsdenii

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yeasts. In Encyclopedia of Animal Science (Pond, W.G., ed), pp. 376–378, CRC

Press.

39. Klieve, A.V. et al. (2003) Establishing populations ofMegasphaera elsdenii YE 34

and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets.

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1200387

BiographiesDr Rosalind Gilbert is a scientist with the Queensland

Department of Agriculture, Fisheries and Forestry, Rumen Ecology

Unit. Her research interests include rumen microbiology and the

role of phages in controlling rumen microbial populations.

Ms Diane Ouwerkerk is a Senior Molecular Biologist within the

Rumen Ecology Unit of the Department of Agriculture, Fisheries

and Forestry based at the Ecosciences Precinct, Dutton Park,

Queensland. Her research interests include the use of molecular

techniques to investigate gut microbial ecosystems, particularly in

ruminants.

Athol Klieve is the Associate Professor in Agricultural Microbiology

at the University of Queensland. He has worked in rumen micro-

biology for 30 years and leads the collaborative UQ/DAFF Rumen

Ecology Unit.



Polymicrobial nature of chronic oral disease

Stuart DashperA, Helen MitchellA, Geoff AdamsA and Eric ReynoldsA,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]

Recentmicrobiome studies have shown that the human oral

microbiome is composed of over 260 abundant bacterial

species that predominantly live as polymicrobial biofilms

accreted to the non-shedding hard surfaces of the teeth. In

addition representatives of both Archaea and Fungi are

found in the oral cavity and there is considerable colonisa-

tionof the soft tissues of themouth.Most of these species are

commensal and form complex biofilm communities that

restrict the colonisation of the oral cavity by exogenous

bacteria. Changes in the polymicrobial biofilm microenvi-

ronment such as those resulting from the effects of chronic

inflammation for subgingival plaque, can lead to the emer-

gence of opportunistic pathogens resulting in dysbiosis and

the development of chronic diseases such as periodontitis in

a susceptiblehost. Theapplicationofmicrobiomic studies to

the analysis of these complex and dynamic communities in

rigorously designed human clinical studies will provide

valuable mechanistic insight into the bacterial succession

and complex interactions involved in the development of

dysbiosis and disease.

The human oral cavity is the entry point of the gastrointestinal tract

and offers a number of microenvironments that enable the prolif-

eration of a wide range of largely commensal bacteria, the vast

majority of which are endemic to the human oral cavity. Consider-

able effort has been expended to identify the approximately 700

prokaryote species that compose the total human oral microbiome.

Over one-third of these species remain uncultivated and less than

half are officially named; however, draft genomes for approximately

half of these taxa are now available from the Human Oral Microbial

Database (www.homd.org/)1.

16S rRNA gene sequence surveys are providing a cost effective

means of studying microbiomes, identifying and enumerating a

relatively unbiased set of the prokaryotic species present, including

uncultivable species. This technique has been adopted for studying

the oral microbiota; however, the results produced have not yet

been definitive, with some studies finding huge variation across

individuals and limited or no differences between healthy and

diseased states. Many factors of the design and analysis of these

experiments can make it difficult to compare results between the

different studies, including pooling of samples, DNA extraction

method, marker gene or region used, primer sets, PCR conditions,

sequencing platform, choice of taxonomic classifier and level of

classification, clustering of read data into microbial groups, and the

statistical methods used for diversity analysis2. The composition of

the healthy oral microbiota can certainly vary considerably across

sites within the mouth, at the same site over time, and from person

to person3,4. Although the 16S rRNA survey techniques are available

most clinical studies to date have been cross-sectional and have

investigated a limited number of bacterial species using either real

time PCR, checkerboard DNA-DNA hybridisation, or more recently,

the Human Oral Microbe Identification Microarray (HOMIM)

techniques. To compound these limitations samples taken from a

limited number of sites within the mouth are often pooled which

can obscure comparative results and is not recommended for

Under theMicroscope


diverse communities such as the oral microbiome. Generating

less information from a larger number of samples is much more

informative than generatingmore information from a small number

of samples, particularly for the classification of diseased and healthy

states5. Furthermore, clinical sampling techniquesmay result inonly

part of the polymicrobial biofilm being collected; with the surface

of the biofilm most closely associated with the host and disease

process poorly represented in the sample.

Chronic periodontitis is an inflammatory disease of the supporting

tissues of the teeth with an endogenous polymicrobial aetiology.

Over 47% of Americans over 30 (64.7million adults) have chronic

periodontitis, distributed as 8.7% mild, 30.0% moderate and 8.5%

severe6. The prevalence and severity of periodontitis increases with

age, with more than 64% of adults aged 65 and over likely to have

moderate or severe periodontitis. Numerous cross-sectional and

longitudinal epidemiological studies have shown associations be-

tween periodontal diseases and a greater risk of certain systemic

diseases and disorders, such as cardiovascular diseases, diabetes,

chronic kidney disease, metabolic syndrome, obesity, rheumatoid

arthritis, Alzheimer’s disease, pre-term andunderweight births, and

some cancers, particularly pancreatic, head and neck and oesopha-

geal cancers. These associations remain even after adjustment for

medical and socio-economic confounding factors.

The concepts of the roles of particular oral bacterial species in

chronic periodontitis have changed over the past two decades but

there is wide consensus that anaerobic, proteolytic, amino acid

fermenting species including Porphyromonas gingivalis, Trepone-

ma denticola and Tannerella forsythia play a crucial role in

initiation and/or progression of disease. A process of bacterial

succession in subgingival plaque has been described where mild

inflammationof thegingival tissue (gingivitis) and theestablishment

of an appropriate microenvironment by early colonising bacteria

allows late colonisers to emerge as opportunistic pathogens7,8.

More recently P. gingivalis has been proposed to be a keystone

pathogen that perturbs the ecological balance enabling the prolif-

eration of other oral bacterial species resulting in the formation of a

dysbiotic polymicrobial plaque, whilst remaining at very low levels

itself9. The keystone pathogen concept was adapted from conser-

vation biology’s keystone species defining a low abundance species

which has a disproportionately large effect on its environment.

While this theory does help explain the major role of a few species

in a complex polymicrobial biofilm, it is not entirely consistent

with prospective clinical trial data demonstrating that this

bacterium, amongst others, proliferates during disease and can

represent a significant proportion of subgingival plaque bacteria at

diseased sites. Furthermore the imminent progression of chronic

periodontitis at a site has been predicted by increases in the relative

proportions of P. gingivalis and/or T. denticola in subgingival

plaque at that site above threshold levels of 10–15%10. This and

more recent research demonstrating mutualism and synergistic

virulence in animal models of periodontitis of multiple late colonis-

ing species found closely associated with disease progression in

humans has led to the more generally supported view of the

emergence of an opportunistic pathogenic polymicrobial biofilm

as the trigger for disease progression in susceptible individuals

(Figure 1)11. A large number of disease-associated species have

been identified, consistent with the dysbiosis-hypothesis12, al-

though further studies are required to differentiate commensal

species that benefit from the disease process as opposed to those

that actually cause the disease.

In addition to the composition of the subgingival plaque polymi-

crobial biofilm, which will be revealed by microbiomic analyses, its

architecture is important as it has been shown that the late colonis-

ing opportunistic periodontal pathogens are found as microcolo-

nies in the outer layer of the biofilm adjacent to the epitheliumof an

inflamed periodontal pocket13. These findings indicate that the

disproportionately large impact of the late colonising opportunistic

pathogens in disease may be explained by their close proximity to

the inflamed tissue and resorbing alveolar bone.

Chronic periodontitis is episodic in nature with acute exacerbations

of destruction followed by periods of dormancy. Currently, diag-

nosis of periodontitis is achieved retrospectively by clinical assess-

ment of attachment loss. This loss of attachment is a result of

pathogenic events that have already occurred at the diseased site,

and any sampling at the time of diagnosis, may fail to identify those

species involved in active destruction. In addition different teeth

within the same patient, as well as different sites around the same

Dysbiosis

Homeostasis

Stable

Oral Healthcrc

Commensal Biofilm Reduced Inflammation

Inflammation

Progressive

Chronic Periodontitis

Pathogenic Biofilm

Figure 1. The changes in the subgingival plaque microbiome and theintimate association with the host inflammatory response that results ina shift from a stable site to one that is undergoing disease progression.

Under theMicroscope


tooth can display varying degrees of disease severity, all undergoing

periodontal disease progression at different rates. Therefore to

conclusively determine the polymicrobial aetiology of chronic peri-

odontitis and how opportunistic pathogens emerge and proliferate

rigorously designed prospective human clinical trials coupled with

microbiomic analyses are essential, followed by the testing of the

bacterial species and communities identified in appropriate in vitro

and animal models to determine their potential as polymicrobial

biofilms to induce dysbiosis and disease.

References1. Dewhirst, F.E. et al. (2010) The human oral microbiome. J. Bacteriol. 192,

5002–5017. doi:10.1128/JB.00542-10

2. Goodrich, J.K. et al. (2014) Conducting a microbiome study. Cell 158, 250–262.

doi:10.1016/j.cell.2014.06.037

3. Simón-Soro, Á. et al. (2013) Microbial geography of the oral cavity. J. Dent. Res.

92, 616–621. doi:10.1177/0022034513488119

4. Ge, X. et al. (2013) Oral microbiome of deep and shallow dental pockets in

chronic periodontitis. PLoS ONE 8, e65520. doi:10.1371/journal.pone.0065520

5. Hamady, M. and Knight, R. (2009) Microbial community profiling for human

microbiome projects: tools, techniques, and challenges. Genome Res. 19,

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

7. Socransky, S.S. et al. (1998) Microbial complexes in subgingival plaque. J. Clin.

Periodontol. 25, 134–144. doi:10.1111/j.1600-051X.1998.tb02419.x

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/

nrmicro2381

9. Hajishengallis, G. et al. (2011) Low-abundance biofilm species orchestrates

inflammatory periodontal disease through the commensal microbiota and com-

plement. Cell Host Microbe 10, 497–506. doi:10.1016/j.chom.2011.10.006

10. Byrne, S.J. et al. (2009) Progression of chronic periodontitis can be predicted

by the levels of Porphyromonas gingivalis and Treponema denticola in sub-

gingival plaque.OralMicrobiol. Immunol.24, 469–477. doi:10.1111/j.1399-302X.

2009.00544.x

11. Tan, K.H. et al. (2014) Porphyromonas gingivalis and Treponema denticola

exhibit metabolic symbioses. PLoS Pathog. 10, e1003955. doi:10.1371/journal.

ppat.1003955

12. Curtis, M.A. (2014) Periodontal microbiology—The lid’s off the box again. J. Dent.

Res. 93, 840–842. doi:10.1177/0022034514542469

13. Zijnge, V. et al. (2010) Oral biofilm architecture on natural teeth. PLoS ONE 5,

e9321. doi:10.1371/journal.pone.0009321

Biographies

Stuart Dashper is a Professor in the Oral Health Cooperative

Research Centre and TheMelbourne Dental School, The University

of Melbourne. Over the past 15 years he has developed a systems

biology approach to the study of chronic oral diseases that incor-

porates the identification and characterisation of bacterial patho-

bionts, the composition and structures of the polymicrobial biofilm

communities in which they dwell, themolecular characterisation of

virulence-related traits and their interactionswithothermembers of

the bacterial community and the host.

Helen Mitchell is a researcher with the Oral Health CRC and

Masters of Science student in Bioinformatics at The University of

Melbourne. She is undertaking comparative genomics of the peri-

odontal pathobiont Porphyromonas gingivalis to determine viru-

lence characteristics and assist in vaccine development. She is using

Next-Generation Sequencing techniques to determine bacterial

biomarkers of early childhood caries in saliva.

Geoff Adams has over 30 years’ experience as a biostatistician

and epidemiologist involved in consulting, teaching, and research.

He has been employed by the Melbourne Dental School and the

Oral Health CRC as a biostatistician and epidemiologist since 1999.

Geoff manages the Oral and Systemic Disease program in the

Oral Health CRC, which is investigating associations between peri-

odontal disease and various systemic conditions.

Eric Reynolds AO PhD FICD FTSE FRACDS is a Melbourne

Laureate Professor and CEO and Director of Research of the Oral

Health CRC. He is also Head of the Oral Biology Section of the

Melbourne Dental School. He has been researching and teaching

for over 30 years on the aetiology and prevention of the two major

oral diseases, dental caries and periodontal diseases, which are

associated with polymicrobial biofilms.

Under theMicroscope


Gastrointestinal microbiota, diet and brainfunctioning

Shakuntla Gondalia

Centre for HumanPsychopharmacology, School ofHealth Science, SwinburneUniversity of Technology, Hawthorn,Vic. 3122, AustraliaTel: +61 3 9214 5100Email: [email protected]

Andrew Scholey

Centre for HumanPsychopharmacology, School ofHealth Science, SwinburneUniversity of Technology, Hawthorn,Vic. 3122, Australia

A growing interest for research in the relationship between

the gastrointestine (GI), GImicrobiota, health and disease is

due to the potential for research identifying intervention

strategies.Preclinical andclinical studieshave indicated that

initial colonisation of bacteria in the GI tract can affect the

individual’s health condition in later life. Diet is an influen-

tial factor in modulating this complex ecosystem and con-

sequently can help to modulate physiological conditions.

The broader role of the GI microbiota in modulation of

pathology and physiology of various diseases has pointed

to the importance of bidirectional communication between

thebrain and theGImicrobiota inmaintaininghomeostasis.

An association of diet withmetabolic diseases is well known

andtherearedietary supplements reported to improvebrain

function and cognitive decline. In addition to the plausible

mechanisms of inflammation and oxidative stress for psy-

chological conditions, more research into the role of the GI

microbiota in combination with dietary factors as a compo-

nent in psychological condition is warranted. From this

work, targeted interventions could result.

Although a link between GI function and health or disease in an

individual hasbeendetermined, theunderlyingmechanismsarenot

clearly understood. TheGI tract of a newborn is rapidly colonised by

microbiota during the birth process through maternal contact and

from the surrounding environment. This microbial ecosystem sta-

bilises in first 2–3 years of life and reaches maturity in the human

adult1. By adulthood, the intestine contains approximately 1012

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

estimated that an adult GI tract harbours between 500 to 1000

different species3 from three major bacterial phyla, Bacteroidetes

(Gram-negative), Firmicutes (Gram-positive) and Actinobacteria

(Gram-positive). The proportion of these phyla in any individual

depends upon that individual’s genetic makeup, dietary habits and

surrounding environment.

The initial inoculation then colonisation of the GI impacts the

GI microbiota throughout life and the dynamic microbial

ecosystem is highly influenced by the surrounding environment

and dietary factors. Any modifications in this highly organised and

complex ecosystem have the potential to influence the normal

physiological functions and are suspected to play a role in obesity,

fatty liver disease, inflammation, diabetes and also psychological

conditions4–8.

Diet that impacts GI microbiota: from infancy

to elderly

Upon ageing, weakening of dentition, salivary function, digestion

and intestinal transit time may affect the intestinal microbiota9,10.

One manageable environmental factor is diet, which has been

shown to significantly impact the GI microbial composition. The

types of bacteria present are dependent on the type of substrates

available, with some bacteria prospering on a specific substrate

while others are unable to utilise that compound11. This selective

substrate usage therefore enablesmodulationof the compositionof

GI microbiota via the diet, which can occur at any stage of life.

However, diet in infancy can greatly affect thematuration process of

the microbial profile, gastrointestinal health and immune system.

Human milk is normally the first dietary exposure to an infant. This

is fermented in the colon, stimulating the growth of specific bacteria

Under theMicroscope


(including Gram positive Bifidobacterium spp.). Infants fed with

breast milk and those on milk formula developed and harboured

different and diverse bacterial populations in their GI tract12. More-

over, the composition of different milk formula facilitates different

types of bacterial colonisation. Infants fed with oligosaccharide

enriched formula harbour higher counts of Bifidobacterium

spp. and Lactobacillus spp. compared to infants fed on unsupple-

mented formulae13. This prebiotic effect of infant food could be of a

major concern as it plays a role in shaping the GI microbiota that is

believed to influence an individual’s health for their entire life. A

significant shift in the GI microbial ecology occurs when infants

switch to a more solid and varied diet, including substantial reduc-

tion in the percentage of Bifidobacterium spp. and Lactobacillus

spp. in the totalmicrobiota14. Throughout adulthoodGImicrobiota

appears to becomemore stable15 as withmore stable dietary habits.

The final shift in composition and function of gutmicrobiota occurs

during theolder age of lifespan. In general, aging is associatedwith a

decline in physiological function including in the immune system

and metabolism that consequently affects the microbial composi-

tion, or vice versa. Human age-related changes reported in the GI

microbial composition include a numerical decrease in Bifidobac-

teruim spp. and Lactobacillus spp. and an increase in Enterobac-

teriaceae and obligate anaerobes such as Clostridium spp.16

Numerous dietary components have been identified as having

positive or negative effect on brain function and behaviour, this

effect can be improved byGImicrobiota. Animal studies andhuman

clinical trials have well demonstrated the role of omega-3 polyun-

saturated fatty acid in normal brain functioning. Administration

of Bifidobacterium spp. in combination with a substrate for eico-

sapentaenoic acid (EPA), alpha-linolenic acid results in increased

concentration of EPA in liver tissue and docosahexaenoic acid

in brain tissues17,18. In wild mice model, probiotics such as

L. helveticus prevented high fat diet-induced anxiety-like behav-

iour19. Diet can modulate the GI microbial composition by provid-

ing a favourable environment while the contrary is also true that

GI microbiota can modulate the effect of diet and in turn host

physiological and psychological function.

Activity of GI microbiota

The GI microbiota can produce a vast range of metabolites and/or

structural components whose generation depends on the availabil-

ity of nutrients and the luminal environment. Thesemetabolites are

subsequently taken up by GI tissues, potentially reach circulation

and other distant tissues and can be excreted in urine and breath. GI

microbiotahavebeen linked tovery importanthealth functions such

as development and role of the immune system7, resistance to

infection by preventing pathogen colonisation20, bioactivation of

beneficial constituents such as polyphenols21, detoxification of

xenobiotics and metabolism of luminal components leading to

formation of a variety of metabolites such as short chain fatty acids

(SCFA), vitamins and several gases11,22,23.

SCFAs such as n-butyrate, acetate and propionate act as key sources

of energy for tissues andpromote cellularmechanisms thatmaintain

tissue integrity. When SCFAs reach the circulatory system, they

impact immune function and inflammation8. SCFAs are also in-

volved in host-microbe signalling and control of colonic pH with

subsequent effects onmicrobial composition, intestinalmotility and

epithelial cell proliferation24. Microbes have also been involved in

enzymatic degradation of complex substrates particularly many

forms of polysaccharides from ingested food25. For example Bac-

teroides thetaiotamicron, produces an array of enzymes for carbo-

hydrate breakdown26. During metabolism some gases such as

methane, hydrogen, hydrogen sulfide and carbon dioxide are

produced within the GI tract. Excess production of these gases may

causeGI problems such as bloating and pain. These gasesmay serve

useful purposes however, it is debatable whether hydrogen sulfide

for example, is largely beneficial or harmful!27.

Furthermore, GI microbiota can influence behaviour and brain

function by influencing the expression of certain body chemicals

such as hormons, neurotransmitters and neurotrophic factors.

Commensal bacteria such as Bifidobacteria infantis can modulate

tryptophan metabolism, this suggest that GI microbiota may influ-

ence the precursor pool for serotonin (5-HT)28. Animal studies have

demonstrated thatGImicrobioa can alsomodulates brain chemistry

such as Brain-DerivedNeurotropic Factor (BDNF) expression in the

hypothalamus and the brainstem29. BDNF is crucially involved in

neurogenesis, brain development and neural circuit formation.

BDNF has also been recognised as an important antiobesity factor.

GI microbiota maintain communication with the host using meta-

bolic, neural, immune and endocrine pathways. For health, homeo-

stasis between the GI microbiota and the host system is essential,

since any imbalance in this arrangement may result in a disease

condition4,5,7.

Interaction between GI microbiota and brain

functioning

The evidence is increasing for a bidirectional route of communica-

tion between brain, gut and GI microbiota which use immune,

neural and endocrine pathways and by this means influence gut-

brain communication, brain function and even humanbehaviour4,5.

The top-down mechanism of the effect of stress and psychological

condition on the GI functions is known from extensive research, in

Under theMicroscope


particular with Inflammatory Bowel Disease, Irritable Bowel Syn-

drome and Crohn’s disease. However, the role of GI microbiota in

brain functions such as stress, cognition and mood need to be

explored more comprehensively.

The GI microbiota influences the release of the major neurotrans-

mitters tryptophan, serotonin, endocannabinoid ligands, ghrelin

and cholecystokinin, which can influence food intake, energy

balance and some brain tasks such as emotion, cognition andmotor

functions28,30. Changes in microbial composition and metabolism

correlate with the concept of ‘inflamm-ageing’, a low-grade chronic

pro-inflammatory status as a common basis for a broad spectrum of

age associated pathologies including cognitive decline and immu-

nosenescence31. Age-associated changes may increase intestinal

permeability and ease the passage of bacterial lipopolysaccharides

(LPS) into circulation, resulting in an elevated systemic LPS level.

When LPS binds to pattern recognition receptors such as toll-like

receptor 4 on immune cells, there is induction of inflammation by

production and release of cytokines, leukotrienes and prostaglan-

din32. Animal studieswithprobiotic supplementationdemonstrated

that probiotics can normalise immune responses, reverse beha-

vioural deficits and restore basal noradrenaline level in response to

stress6. The probiotics also normalise central nervous system (CNS)

biochemistry and improve behaviour in a mouse model of colitis,

through vagal nerve pathways for gut-brain communication33. Psy-

chological responses to the GI microbiome composition may be an

important factor in understanding the increasing prevalence of

psychological conditions in the community and research into this

topic should be promoted.

Conclusion

Clinically, a psychological conditiondoesnot standalone since there

are frequently immune system and GI comorbidities. Antidepres-

sants are the commonest treatment, and as such are focused on a

top-down approach. However, growing evidence from increasing

numbers of animal studies and human trials suggest that the GI

microbiota composition can be correlated with the incidence of

complex conditions such as cognitive decline, anxiety and depres-

sion. Detailed clinical interpretation is warranted so that novel

interventions in neuropsychological conditions can be employed.

Preclinical research combining detailed exploration of theGImicro-

biota in well-designed human cohort studies investigating the

impact of antibiotics, probiotics and diet on the brain and CNS

functions will inform us of the importance of bottom-up mechan-

isms in neuropsychological conditions. Further research in this

emerging area will provide novel targets for interventions in psy-

chological disorders.

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Under theMicroscope


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BiographiesShakuntla Gondalia is as Postdoctoral Fellow at Swinburne Uni-

versity of Technology, Victoria and her research interest incorpo-

rates gastrointestinal microbial ecology and nutritional intervention

with thepotential to improve thehealth and cognitive performance.

Her research aims to better understand the effectivemechanisms of

GI microbiota, nutritional and dietary intervention on individual’s

physiological and psychological health condition.

Professor Andrew Scholey is director of the Centre for Human

Psychopharmacology at Swinburne University, Melbourne. He is a

leading international researcher into the neurocognitive effects of

natural products, supplements and food components, having pub-

lished over 160 peer-reviewed journal articles, books and book

chapters. Scholey has been lead investigator in a series of studies

into the humanbiobehavioural effects of natural products, and their

neurocognition-enhancing and anti-stress/anxiolytic properties

(including first-into human neurocognitive assessment of Ginseng,

Sage and Lemon balm among others). His current research focuses

on neuroimaging and biomarker techniques to better understand

the mechanisms of cognitive enhancement.

Under theMicroscope


Marsupial oral cavity microbiome

Philip S BirdA,D, Wayne SJ BoardmanB, Darren J TrottB and Linda L BlackallC

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

disease within their lifetimes and include a large number of

endogenous cariogenic, periodontal and other opportunis-

tic pathogens. Studies over many decades have attempted

to determine which bacteria are involved in oral diseases.

Earlier studiesusedexclusively culture-basedmethods.Now

culture-independent methods are being used to determine

the composition of the microbiome in health and disease.

Therehavebeen limitednumbersof studies of themarsupial

microbiome and this report covers some of the research of

those studies.

Dental plaque, a natural oral biofilm is composed of many diverse

bacterial species, some of which are involved in the aetiology of

periodontal diseases (gingivitis and periodontitis)1. Factors such as

indigenous bacteria, host immune system, diet, host susceptibility

and time, interplay in thesediseases2. Therehavebeenmany studies

determining which of the causative agent(s) initiate oral diseases in

humans and domesticated animals. Marsupials also have oral dis-

eases3,4 and culture-dependent studies have shown a range of

bacteria can be isolated from the marsupial oral cavity5. However

culture-based studies, while useful to enable precise characterisa-

tion of putative periodontopathogens, generally underestimate

microbial community diversity. Culture-independent methods,

such as high-throughput DNA sequencing reveal a rich and diverse

bacterial community in the oral cavities of humans and companion

animals6-8, and the marsupial microbiome should also be more

thoroughly studied too.

The first bacteria ever to be seen under a microscope were the

plaque bacteria taken from Antonie van Leeuwenhoek’s own teeth

and reported in a letter to the Royal Society on 17 September 1683.

These first recorded observations of living bacteria showed ‘an

unbelievable great company of living animalcules and of enormous

number of a variety of shapes and sizes’. Since then many oral

prokaryotic species have been described. The Human Oral Micro-

biome Database (www.homd.org/) has to-date >200 bacterial spe-

cies described fromculture-dependentmethods and approximately

1,000 phylotypes detected by 16S rRNA gene sequencing of oral

samples from the human oral cavity using culture-independent

methods9. A search of the International Journal of Systematic and

EvolutionaryMicrobiologyover thepast 10 years for ‘oral’ revealed

a host of novel bacterial species isolated from the oral cavity of

humans and animals, including domesticated andwild (free-ranging

and captive) animals. In marsupials, novel microbial species were

revealed in the oral cavity of macropods associated with gingivitis

and oral necrobacillosis10.

In domesticated and wild animals, using culture-dependent meth-

ods, associations with disease and specific bacterial species have

been reported5. More recently, researchers have used high-

throughput DNA sequencing to study the oral microbiota of

healthy cats8 and dogs7. In adult horses 67% of 203 operational

taxonomic units (OTUs) were recovered, with the most frequent

genera being Prevotella and Porphyromonas (T Chinkangsadarn,

GJWilson, SWCorley and PS Bird, 2014, unpublished). In each case,

the results revealed a rich and diverse bacterial community in

much higher numbers than identified using culture- and cloning-

based studies. Therefore, in studies of animals with known oral

health status, health and disease could be correlated with the oral

microbiota detected using these new technologies.

Under theMicroscope


We have shown that oral diseases such as gingivitis and periodontitis

can be found in a range of native Australian animals including

macropods, koalas (Phascolarctos cinereus), brushtail possums

(Trichosurus vulpecular) and bandicoots (Isoodon macrourus),

and that black-pigmented, anaerobic bacteria, belonging to the

genera Porphyromonas and Prevotella, are part of themicrobiota5.

Earlier studiesusingculturedependentmethods, showed that in the

normal oral microbiota of macropods, Gram-negative anaerobes

were poorly represented11. In contrast, Dent and co-workers12

reported that macropod oral cavities had a noticeable predomi-

nance of Gram-negative andGram-positive rods with the facultative

anaerobic Gram-negative rods comprising 40% of the cultivable

organisms although no Bacteroides spp. were isolated. In a study of

10 species of kangaroos and wallabies, black-pigmented anaerobic

bacteria comprised 21% of their normal oral microbiota13. Thus the

early culture-based studies have shown variability in the microbes

isolated and suggest that this approach has limitations.

Koalas do present with severe periodontal disease4 and with severe

loss of alveolar boneassociatedwith age andconditions suchas food

impaction3 (Figure 1). In koalas <7 years old with good oral health,

there was an absence of black-pigmented bacteria, compared to

koalas >7 years of age, where 50% harboured black-pigmented

bacteria, the majority of which were identified as Porphyromonas

gingivalis-like14. Currentwork characterising this bacterium, shows

the organism to be novel and that it may be associated with

periodontal disease in marsupials [Bird et al, 2015, submitted].

Another intriguingquestion relates to thekoala’sdietwhichconsists

of Eucalyptus leaves. How have the oral bacteria evolved in the

presence of such a toxic diet (e.g. high in essential oils) and how

does this affect the koala’s oral microbiome?

Oral necrobacillosis or lumpy jaw as it is commonly known, is a

leading cause of mortality in captive macropods and has been

reported in free-rangingmacropods15. The disease progresses from

plaque formation, gingivitis and periodontal disease to a necrotis-

ing, fatal osteomyelitis,16 (Figure 2) with all macropods susceptible,

particularly Eastern grey kangaroos17. Early studies of macropods

with lumpy jaw showed that Fusobacterium necrophorumwas the

most frequent isolate from lesions (81% prevalence) as well as the

most abundant organism in mixed cultures. It also was isolated in

high abundance from gingival margin samples taken from sites

remote from the lumpy jaw lesions in 61% of the animals with

disease18. While the principle infective agent in Australia appears to

be F. necrophorum, other organisms appear to play a role in this

disease. Recent work has shown that F. necrophorum sub-species

necrophorum is associated with organisms resembling Porphyro-

monas gulae in lumpy jaw inmacropods in South Australian zoos19.

Porphyromonas organisms distinct from both P. gingivalis and

P. gulae have been proposed as a novel species14 and were isolated

with increasing frequency from the oral cavity of macropods in our

studies, whichwarrants further evaluation into the role of this newly

described organism in jaw disease.

A study of the tammar wallaby pouch young suggested factors that

protect young animals against potentially pathogenic microbial

infections could include themicrobiome from thematernal saliva20.

The microbiomes of the pouch and saliva from the mother were

compared with the gastrointestinal tract (GIT) microbiome of the

pouch young using 16S rRNA gene comparative methods. Each

study site had a unique microbiome20. The maternal pouch har-

boured 41 unique Actinobacteria phylotypes, while in the saliva

there were 48 unique Proteobacteria phylotypes. The GIT of the

pouch young had a complex microbiome of 53 unique phylotypes

andof these,only nineweredetectedat eithermaternal site.Overall,

themajority of bacteria detected were novel species and each study

site possessed its own unique microbiome20.

In conclusion, a number of culture-based studies on the oral

microbiota of marsupials were conducted some 20–30 years ago.

Nowwith the deep sequencing culture-independentmethods it will

be possible to detect novel bacterial species in their oral cavities

and these are likely to have unique properties. In addition, new

studies will undoubtedly lead to insights into their evolution,

diversity and ecological role. We can speculate that co-evolution

of the marsupial oral microbiome has occurred with its host and

organisms such as the newly identified porphyromonad unique

to marsupials may represent an ancestral lineage distinct from

P. gulae and P. gingivalis. The oral microbiome of our marsupials

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 MPettett).

Under theMicroscope


thorough exploration by keen and dedicated microbiologists for

novel bacteria and associated oral diseases.

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5002–5017. doi:10.1128/JB.00542-10

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/

j.rvsc.2013.08.010

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

45770560083101

12. Dent, V.E. (1979) The bacteriology of dental plaque from a variety of zoo-

maintained mammalian species. Arch. Oral Biol. 24, 277–282. doi:10.1016/

0003-9969(79)90089-X

13. Samuel, J.L. (1982) The normal flora of the mouths of macropods (Marsupialia:

macropodidae).Arch. Oral Biol.27, 141–146. doi:10.1016/0003-9969(82)90134-0

14. Mikkelsen, D. et al. (2008) Phylogenetic analysis of Porphyromonas species

isolated from the oral cavity of Australian marsupials. Environ. Microbiol. 10,

2425–2432. doi:10.1111/j.1462-2920.2008.01668.x

15. Borland, D. et al. (2012) Oral necrobacillosis (‘lumpy jaw’) in a free-ranging

population of eastern grey kangaroos (Macropus giganteus) in Victoria. Aust.

Mammal. 34, 29–35. doi:10.1071/AM10031

16. Bakal-Weiss, M. et al. (2010) Use of a sustained release chlorhexidine varnish

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.

In Fourth International Conference on the Wildlife Diseases Association.

Sydney, Australia.

18. Samuel, J.L. (1983) Jaw disease in macropod marsupials: bacterial flora isolated

from lesions and from themouths of affected animals. Vet. Microbiol. 8, 373–387.

doi:10.1016/0378-1135(83)90050-0

19. Antiabong, J.F. et al. (2013) Amolecular survey of a captive wallaby population for

periodontopathogens and the co-incidence of Fusobacterium necrophorum

subspecies necrophorum with periodontal diseases. Vet. Microbiol. 163,

335–343. doi:10.1016/j.vetmic.2013.01.012

20. Chhour, K.L. et al. (2010) An observational study of the microbiome of the

maternal pouch and saliva of the tammar wallaby, Macropus eugenii, and of

the gastrointestinal tract of the pouch young. Microbiology 156, 798–808.

doi:10.1099/mic.0.031997-0

BiographiesDr Philip Bird is an Adjunct Associate Professor at the School of

Veterinary Science. His research interests are the oral microbiology

of human and animals, especially that of native Australian animals.

Dr Wayne Boardman is Senior Lecturer, at the School of Animal

and Veterinary Sciences, University of Adelaide. An experienced

wildlife veterinarian, he is a diplomate of the European College of

Zoological Medicine in Wildlife Population Health.

Dr Darren Trott is Associate Professor of Veterinary Microbiology

at The University of Adelaide. His research interests cover compar-

ative aspects of antimicrobial resistance in animals andhumans, new

drug development and gastrointestinal microbial ecology.

Linda L Blackall is a microbial ecologist who has studied many

different complex microbial communities ranging from host asso-

ciated through to free living in numerous environments. Her

research has covered mammalian microbiomes spanning marsu-

pials, humans, ruminants and horses and the methods used allow

elucidation of massive microbial complexity and function in these

diverse biomes. She is a Professor of Biosciences at Swinburne

University of Technology in the Faculty of Science, Engineering and

Technology.

Figure 2. Wallaby skull showing swollen lower jaw due to oral necrobacillosis or lumpy jaw.

Under theMicroscope


Relative abundance of Mycobacterium in ovineJohne’s disease

Nostudyhasdeterminedwhat proportion of the totalmicro-

biota comprises the genusMycobacterium in ovine Johne’s

disease (OJD) tissues. We aimed to assess the relative abun-

dance of Mycobacterium in the ileocaecal lymph node,

involved and uninvolved ileal mucosa from sheep with and

without OJD, using three extraction methods. Eight sheep,

fourwithand fourwithoutOJD,were recruited.Pyrosequen-

cingof the16SrRNAgene amplicons for all samples revealed

that Mycobacterium represented between 0-92% (average

38%) of the total microbiota of samples from sheep with

OJD, and 0-85% (average 13%) of sheep without OJD. Only

sheepwithOJDhad samples that were positive for the IS900

(MAP) element. Mycobacterial strains other than MAP may

provide competitive exclusion ofMAP and should be further

investigated.

Mycobacterium avium subspecies paratuberculosis (MAP) is

the etiologic agent of ovine Johne’s disease (OJD), a chronic,

Andy O LeuA, Paul PavliB,C, David M GordonA, Jeff CaveD, Jacek M GowzdzE, Nick LindenD,Grant Rawlin

E, Gwen E Allison

A,Cand Claire L O’Brien

A,B,C,F

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]

LabReport


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 com-

prisesMycobacterium. Cheung et al. (2013)2 found thatMycobac-

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 commu-

nity in all infectious states andspecimens?MAP is aputative triggerof

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?

While other studies have quantified Mycobacterium or MAP in

clinical and subclinical specimens4,5, no study has determined what

fraction of the microbial community comprises Mycobacterium in

mucosa from sheep with and without OJD. The primary aim is to

assess the relative abundance of Mycobacterium in the microbial

communities of macroscopically normal bowel mucosa obtained

>50 cm downstream the ileocaecal valve (sheep without OJD)

or involved mucosa (sheep with OJD), involved (or normal for

sheep without OJD) ileal mucosa adjacent the ileocaecal lymph

node, and the ileocaecal lymph node of sheep with and without

OJD.

Samples of involved and uninvolved ileal mucosa, and ileocaecal

lymph nodes were collected from eight sheep, four with and four

without OJD, from farms around Rutherglen, Australia. Sterile

gloves and implements were used to extract the ileocaecal node

before the bowel was opened, so that it did not come into contact

with the skin or bowel microbiota. Total DNA was extracted from

each sample using three different extraction methods. The first

protocol followed the Qiagen DNeasy kit protocol and included an

enzymatic lysis step and bead-beating step. The second and third

extractions were the same as the first, except the second protocol

included a freeze-thaw step, and the third protocol a boiling step.

Technical replicates were included for the first protocol. All samples

were amplified using barcoded primers targeting the universal

bacterial16S rRNA gene, and pyrosequenced using a 454 Genome

Sequencer FLX-Titanium platform, as previously described3.

Sequences were analysed using Mothur6. IS900 PCR was used to

determine whether or not Mycobacterium detected in a given

sample was MAP, or not.

Four sheep (1, 2, 4 and 5; for example, see Figure 1) were diagnosed

withOJDbyqualified veterinarians andpathologists, usingmethods

previously described7,8. Briefly, sheep were initially assessed as

having OJD, or not, based on physical signs, including condition,

scouring, ill-thrift, and lethargy during mobility. Diagnosis was

confirmed by a NATA-accredited pathology laboratory (AgriBio,

Victoria), and included MAP culture and DNA analysis, as well as

microscopic visualization of acid-fast bacteria using ZN staining .

Sheep 1 was initially suspected to have OJD, however negative

culture and staining results excluded it from a diagnosis of OJD, and

it was found to have metastatic cancer.

Pyrosequencing showed thatMycobacterium representedbetween

0–92% (average 38%) of the total microbiota of samples from sheep

withOJD,and0-85%(average13%)of sheepwithoutOJD(Figure2).

Mucosal samples from sheep 7were excluded due to the inability to

amplify products from them. Sheep 4 and 5 each had a positive

lymph node sample for one extraction, comprising 6% and 68%

Mycobacterium reads, respectively.These levelswere similar to that

observed in the mucosal samples from the same animal.

The lymph node and mucosa samples of sheep 1 were negative

when tested with theMAP-specific primers. Sheep 1may have had a

paucibacillary form of OJD. Samples from all other sheep with

OJD were positive, except the normal mucosa sample of sheep 4

(Table 1). All samples fromsheepwithoutOJDwere negative for the

IS900 element.

No significant effect of extraction method was detected for either

Simpson or Shannon diversity estimates (Simpson Diversity, AOV:

P > F=0.384; ShannonDiversity, AOV: P > F=0.443), nor technical

replicates (Simpson Diversity, AOV: P > F=0.398; Shannon Diver-

sity, AOV: P > F=0.566).

Each sample was covered by an average of 2, 644 quality sequences.

Six phyla (Firmicutes, Bacteroidetes, Actinobacteria, Proteobac-

teria, Synergistetes, and TM7) represented ~98% of all sequences

Figure 1. Ovine Johne’s disease affected sheep #2, showing physicalsigns of wasting.

LabReport


(Figure 3). Actinobacteria, of which Mycobacterium is a member,

was overrepresented in sheep with OJD, except sheep 1. Microbial

community diversity declined as Mycobacterium increased

(R2=0.705, p=0.001) (Figure 4).

To our knowledge, this is the first study to describe the full

microbial community of mucosa from sheep with and without

OJD. Although studies have enumeratedMAP in the feces of sheep

using RT-PCR5,9, no study has determined what fraction of the

100

90

80

70

60

50

Myc

obac

teriu

m (

%)

40

30

20

10

0

Figure 2. Proportion of involved and normal mucosa and lymph node tissues (sheep 4 and 5) containing Mycobacterium. Sheep with Johne’sdisease are coloured orange, without blue. Technical replicates (normal mucosa only) have diagonal texturing. The first numeral in the labelindicates 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 8did 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

Sex F F F F F F F F

Age 7 4 7 7 4 6 3 6

Vaccination N Y N N Y N Y N

Breed Merino Merino Merino X

Leicester

Merino X

Leicester

Merino Merino Merino Merino

Intestinal

lesions

Severe Moderate None Moderate Mild None None None

MAP culture + + – + + – – –

AFB in

Involved

Mucosa

Absent Occasional Absent Numerous Numerous Absent Absent Absent

AFB in

Lymph node

Absent Absent Absent Moderate Numerous Absent Absent Absent

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.

LabReport


microbial community Mycobacterium comprises, and whether or

not these proportions differ in sheep with and without OJD, as

well as various tissue types.

Bacterial translocation to the ileocaecal lymphnodewas observed in

50% (2/4) of sheep diagnosed with OJD. The microbial community

of thesenodeswas similar to thatof the involvedandnormalmucosa

from the same sheep. Bacteria in the node are likely to be secondary

invaders, present in the node due to a breakdown in the mucosal

barrier. Using the same methods described here, we found that

bacterial translocation in Crohn’s disease of humans is also non-

specific. We also did not detectMycobacterium in this study using

16S rRNA gene sequencing3. This suggests that Mycobacterium is

unlikely to be a cause of Crohn’s disease.

Samples from sheep without OJD were not positive for MAP

culture or the IS900 element, but other strains of Mycobacterium

were present, representing on average 13% of the total microbiota.

Strains of Mycobacterium other than MAP could occupy a niche

in the sheep gastrointestinal tract that MAP would otherwise

occupy, thereby protecting the animal from colonization of

MAP. Future studies should assess the diversity of Mycobacterium

strains associated with the mucosa of sheep with and without

OJD.

We found the abundance of Mycobacterium, relative to other

bacterial taxa in the mucosal samples, was significantly higher in

sheep with OJD. Increases in Mycobacterium result in large

decreases in Firmicutes. Members of the Firmicutes are important

producers of short-chain fatty acids, which are shown to improve

intestinal barrier function and repress inflammation10. Dysbiosis

and reduced diversity would likely exacerbate symptoms of OJD,

as beneficial bacteria are decreased and their roles in homeostasis

unfulfilled.

Mycobacterium makes up a large fraction of the microbial com-

munities of mucosa from sheep with OJD. Future studies should

determine if MAP is the sole contributor to these levels of Myco-

bacterium in sheep with OJD, and assess the diversity ofMycobac-

terium in sheep with and without OJD. Other Mycobacterium

strains may provide competitive exclusion of MAP.

Acknowledgements

We thankGreg Seymour for assisting in the collection of specimens.

Figure 3. Proportion of the six dominant bacterial phyla in tissues from sheepwith andwithout ovine Johne’s disease (OJD), showing a predominanceof 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.

7

6

5

4

3

Sha

nnon

inde

x

Mycobacterium (%)

2

1

00 20 40 60 80 100

Sheep 1

Sheep 2

Sheep 3

Sheep 4

Sheep 5

Sheep 6

Sheep 8

120

Figure 4. Figure showing a negative correlation between the Shannondiversity index and the proportion of Mycobacterium in the mucosalmicrobiota of sheep. Sheep 1, 2, 4 and 5 were diagnosed with OJD.

LabReport


References1. Salem, M. et al. (2013) Mycobacterium avium subspecies paratuberculosis:

an insidious problem for the ruminant industry. Trop. Anim. Health Prod. 45,

351–366. doi:10.1007/s11250-012-0274-2

2. Cheung, M.K. et al. (2013) Sputummicrobiota in tuberculosis as revealed by 16S

rRNA pyrosequencing. PLoS ONE 8, e54574. doi:10.1371/journal.pone.0054574

3. O’Brien, C.L. et al. (2014) Detection of bacterial DNA in lymph nodes of Crohn’s

disease patients using high throughput sequencing. Gut 63, 1596–1606.

doi:10.1136/gutjnl-2013-305320

4. Park, K.T. et al. (2014) Development of a novel DNA extraction method for

identification and quantification of Mycobacterium avium subsp. paratubercu-

losis from tissue samples by real-time PCR. J. Microbiol. Methods 99, 58–65.

doi:10.1016/j.mimet.2014.02.003

5. Logar, K. et al. (2012) Evaluation of combined high-efficiency DNA extraction and

real-time PCR for detection ofMycobacteriumavium subsp. paratuberculosis in

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

6. Schloss, P.D. et al. (2009) Introducing mothur: open-source, platform-indepen-

dent, community-supported software for describing and comparing microbial

communities. Appl. Environ. Microbiol. 75, 7537–7541. doi:10.1128/

AEM.01541-09

7. Gwozdz, J.M. et al. (2000) Vaccination against paratuberculosis of lambs already

infected experimentally with Mycobacterium avium subspecies paratubercu-

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

(M7H9C) for routine culture ofMycobacterium avium subsp. paratuberculosis

to replace modified Bactec 12B medium. J. Clin. Microbiol. 51, 3993–4000.

doi:10.1128/JCM.01373-13

9. Sting, R. et al. (2014) Detection of Mycobacterium avium subsp. paratubercu-

losis in faeces using different procedures of pre-treatment for real-time PCR in

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

colon. FEMS Microbiol. Lett. 217, 133–139. doi:10.1111/j.1574-6968.2002.

tb11467.x

Biographies

Andy Leu graduated from the Australian National University (ANU)

with aBachelor ofMedical Science (Honours I) in2013.HisHonours

project examined the relative abundanceofMycobacteriumavium.

paratuberculosis (MAP) in Johne’s disease tissues. Andy was re-

cently awarded an Australian Postgraduate Award scholarship to

pursue a PhD under the supervision of Dr Gene Tyson.

Paul Pavli is a gastroenterologist with clinical and basic scientific

research interests in the inflammatory bowel diseases. He was on

the steering committee of the multicentre Australian study using

long-term anti-mycobacterial agents in Crohn’s disease (Selby

et al., Gastroenterology, 2007). Current research interests are in

the interaction between the innate inflammatory response and the

intestinal microbiome.

David Gordon received his BSc from King’s College London and

his PhD fromMcGill University,Montreal.His research concerns the

ecology, population genetics and evolution of the Enterobacter-

iaceae, especially E. coli.

Jeff Cave graduated from Murdoch University in 1988 and spent

twoyears inmixedprivatepractice inSouthAustralia beforeworking

in the Pacific for much of the 1990s. For the past 16 years he has

worked as the District Veterinary Officer in Wodonga, Victoria.

Jacek Gwozdz is a Veterinary Pathologist at the State veterinary

laboratory in Victoria. He received his PhD in Veterinary Science

fromtheMasseyUniversity inNewZealandandworkedover thepast

16 years in research and diagnostic microbiology and pathology.

NickLinden is a research scientistwith theVictorianDepartmentof

Primary Industries and Environment, he has a particular interest in

the lamb supply chain and is passionate about linking on farm

production through to carcass appraisal and consumer acceptance.

His recent projects have been focused on lamb feed conversion

efficiency as well as on-line grading of lamb carcasses.

Grant Rawlin is Research Leader of Veterinary Pathobiology at the

State veterinary laboratory in Victoria. His career has spanned

veterinary diagnostics as a pathologist, research in veterinary and

humanmicrobiologyandresearch intocontrolof infectiousdiseases

of animals at a population level.

Dr Gwen Allison received her PhD in Microbiology from North

Carolina State University. Her research interests have included

molecular biology of lactic acid bacteria and microbial ecology of

the gastrointestinal tract and environment. The current research

was conducted when Dr Allison was a Senior Lecturer at the ANU.

Dr Claire O’Brien received her PhD from the Australian National

University Medical School (ANUMS) in 2012. Her main research

interest is thegutmicrobiome,particularly inCrohn’sdisease.Claire

is an NHMRC Peter Doherty Fellow at the ANUMS and Canberra

Hospital.

LabReport


Interactions with other microbiology societiesthrough Microbiology AustraliaThe September 2014 issue ofMicrobiology Australia was a special

issue that reflected on the ‘Microbial diseases and products that

shaped world history’. A major focus involved microbes of WWI

and their effects on ANZACs and Turkish soldiers. The timing of

the issue is particularly fitting as we reflect on the 100th anniversary

of the terrible losses suffered by both sides involved in the Gallipoli

conflict.

The idea for the issue came from Ipek Kurtböke who Guest

Edited the joint effort between the Australian, New Zealand and

Turkish Microbiology Societies. The issue contains excellent

articles on the major diseases of world history, and early efforts to

control them.

TheTurkish articleswerehighly informative about their experiences

with smallpox, tuberculosis and typhus as well as the huge losses

the Turkish troops suffered at the hands of infectious diseases.

In addition, the articles remind us that much is owed to the Turks

for the early lessons they learned through their contributions to

infection control. This is particularly so for their early adoption of

variolation for the prevention of smallpox, an experience that led

directly to Jenner’s seminalworkwith cowpox and thedevelopment

of modern vaccination.

At the invitation of the TurkishMicrobiology 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. . .Youare now lying in the soil of a friendly country. Therefore restin peace. There is nodifference between the Johnnies and theMehmets to us where they lie side by side here in this countryof ours. . .You, themothers,who sent their sons from farawaycountries wipe away your tears; your sons are now lying inour bosom and are in peace, after having lost their lives onthis land they have become our sons as well.

Wehave learnedmuch fromour interactions. It was a privilege to be

hosted by such gracious hosts and we look forward to welcoming a

delegation of Turkish microbiologists to Canberra for ASM2015.

The success of our interactions with other microbiology societies

is something ASM would like to foster and it is hoped that every

few years a similar joint issue of Microbiology Australia can be

produced.

ASM History SIG: Microbiology AustraliaThe ASM History SIG was launched at ASM Adelaide 2013 at a

meeting on 12 noon Tuesday 9th July 2013 and Dr Diane Lightfoot

was elected convener for the first 2 years of this SIG.

The purpose of this SIG is to record the history of Microbiology in

Australia, in the ASM archives, including details about certain

microbiologists, microbiological institutions, and microorganisms,

as well as disseminate this information to ASM members.

Regularly notes on topics of interest will be contributed to the ASM

Victorian Branch Newsletter from the History SIG (e.g. prominent

microbiologists, significantmicrobiological discoveries by Victorian

microbiologists, microbiological institutions/major events at these

institutions). In future it is intended to have a History of Australian

Microbiology Symposium at the ASM annual scientific meeting.

The AGM of the History SIG will be held at the ASM scientific

meeting in Canberra July 2015.

If ASM members have that significant ASM memorabilia that they

would like to donate to the ASM archives or suggestions of topics

suitable for possible symposia at the ASM Annual Meetings. Please

send details of memorabilia or suggestions/topics of interest or for

possible symposia to:

HistorySIGconvener, c/oAustralianSociety forMicrobiologyOffice,

9/397 Smith Street, Fitzroy, Vic. 3068, Australia.

Nezahat Gurler, Paul Young, Ian Macreadie, _Ipek Kurtboke, PhilippeDesmeth and Bulent Gurler.

ASMAffairs


Recent developments in virology by AustralianresearchersJoseph R Freitas and Suresh Mahalingam

Institute for Glycomics, Griffith University, Gold Coast, Queensland

Virology is a growing field within Australia. Increased funding is

being allocated to discovering how viruses interact with their

host/vectors(s) and to the development of better treatments

and vaccines. There have been many recent exciting new develop-

ments in Australian virology. Space limitations mean that we can

only highlight a small number of these achievements in this brief

overview of the current virology landscape in Australia.

The increasing prevalence or re-emergence of certain alphaviruses,

such as Ross River virus (RRV), Sindbis virus, and Chikungunya

virus, is a cause for concern and has attracted increased research

interest in recent years. These viruses are known as arthritogenic

alphaviruses because they cause arthritis-like symptoms. The ability

of these viruses to directly induce bone pathology has remained

poorly defined to date. Chen et al.1 recently shed some light on this

issue by revealing that RRV can infect human osteoblasts and that

osteoblast infection leads to IL-6-dependent bone loss in a mouse

model. This discovery of the interaction between osteoblasts,

inflammatory factors and alphaviruses is a plausible explanation for

how viruses cause chronic joint pain.

Infections with certain viruses, such as West Nile virus (WNV) and

dengue virus, can lead to encephalitis, which is associated with

high mortality. Inflammatory cells play a central in the progression

of these diseases. Currently there are no specific targeted treat-

ments to modulate the action of inflammatory cells. Getts et al.2

recently demonstrated that infusion of immune-modifying micro-

particles (IMPs) into WNV-infected mice significantly reduced

the symptoms of central nervous system infection. The continued

injection of IMPs to mice over several days led to an improved

survival rate. Similar therapeutic effects were shown for other

inflammation-mediated diseases. The therapeutic potential of

such IMPs for a variety of immune-related disorders looks to be

very promising.

Dengue (DENV) is a mosquito-borne virus that infects hundreds

of millions of people annually. It has a widespread geographical

prevalence that continues to expand. There is an urgent need for an

effective vaccine against DENV. However, this has proven difficult

due to the virus having four different serotypes, with a general

consensus that a successful vaccine will need to induce immunity to

each specific serotype. The identification of antibodies with broad

cross-reactivity to all serotypes by Dejnirattisai et al.3 led to the

discovery of a novel DENV epitope, which has high potential for use

as a successful vaccine antigen. Although this study was performed

outside of Australia, the threat of DENV in Australia and the strong

collaborative input fromUniversity ofMelbourne researchersmerits

its mention here.

Viruses with the ability to pass from animals to humans (‘zoonotic

viruses’) are very unpredictable because they have the potential to

mutate and become deadly in new hosts. The most notable local

example of this is theHendra virus outbreaks that occurred over the

past two decades. Research into this virus and its natural hosts led to

the successful development and deployment of a vaccine against

Hendra in 20124. A newly identified virus found in Australian bats,

dubbed Cedar virus, by Marsh et al.5 in 2012 was found to be very

similar to the Hendra and Nipah viruses. The one key difference is

that it does not cause disease in several mammals that are suscep-

tible to Hendra and Nipah. This fascinating discovery will no doubt

assist scientists to understand what exactly it is that makes certain

viruses deadly and others less so.

Norovirus (NoV) is the leading cause of gastroenteritis worldwide

and is known to cause thousands of deaths in developing countries.

Most outbreaks occur within institutional settings such as hospitals

and aged-care facilities. One particular genotype of the virus is

responsible for the majority of infections, the genogroup II geno-

type 46. Since current treatment for NoV infections is largely

preventative, there is an urgent need for an effective vaccine or

antiviral. By using high throughput screening methods against the

RNA polymerase of NoV, Eltahla et al.7 identified a very promising

target for development of effective antivirals against this disease.

With these and the many other researchers around Australia cur-

rently active in thefield,we canexpect continuingmajor advances in

the discipline of virology in 2015 and in the years to come.

References1. Chen, W. et al. (2014) Arthritogenic alphaviral infection perturbs osteoblast

function and triggers pathologic bone loss. Proc. Natl. Acad. Sci. USA 111,

6040–6045. doi:10.1073/pnas.1318859111

2. Getts, DR et al. (2014) Therapeutic inflammatory monocyte modulation using

immune-modifying microparticles. Sci. Transl. Med. 6, 219ra7.

3. Dejnirattisai, W. et al. (2015) A new class of highly potent, broadly neutralizing

antibodies isolated from viremic patients infected with dengue virus. Nat. Immu-

nol. 16, 170–177. doi:10.1038/ni.3058

4. Broder, C.C. et al. (2013) A treatment for and vaccine against the deadlyHendra and

Nipah viruses. Antiviral Res. 100, 8–13. doi:10.1016/j.antiviral.2013.06.012

5. Marsh, G.A. et al. (2012) Cedar virus: a novel Henipavirus isolated from Australian

bats. PLoS Pathog. 8, e1002836. doi:10.1371/journal.ppat.1002836

6. Eden, J.S. et al. (2013) Recombinationwithin thepandemicNorovirusGII.4 lineage.

J. Virol. 87, 6270–6282. doi:10.1128/JVI.03464-12

7. Eltahla, A.A. et al. (2014) Nonnucleoside inhibitors of Norovirus RNA

polymerase: scaffolds for rational drug design. Antimicrob. Agents Chemother.

58, 3115–3123. doi:10.1128/AAC.02799-13

ASMAffairs


Clinical Serology and Molecular SIGFrom the retiring National Convenor, David Dickeson

The Clinical Serology Special Interest Group was established after a

meeting in Adelaide in 1989with Peter Robertson (now retired from

PrinceofWalesHospital, Randwick) the inaugural national convenor

andonly a fewdozenmembers. Itwas established toprovide a forum

for the discussion of serological techniques with the objective to

develop a strategy to monitor and control the quality of diagnostic

reagents. This was published in 1994 (Backhouse et al., Australian

Microbiologist,1994;15:37–45).BranchesofthisSIGwereformedin

most states from 1989 onwards. I became involved as NSW branch

treasurer in 1993with LeonHeron as convenor and I have remained

as secretary/treasurer ever since. The group was expanded in 2003

to include molecular techniques in the medical diagnostic industry

and the combined SIG convenor was Robyn Wood (formerly from

Queensland Medical Laboratory, then TGA). In 2004 I took on the

position as convenor at the ASM annualmeeting in Sydney and have

continued in that role until now.

The CS&M SIG Working Group was formed in 2009 after several

ASM meetings and NRL workshops with the great assistance of

Sheena Adamson (Communicable Diseases Branch, NSW Health)

who remains as the working group secretary. This group is

overseen by the ASM Clinical Microbiology Standing Committee

and aims to be a proactive source of education and information

and able to give advice on serology and related molecular testing.

Teleconferences held every 3 months have kept an expert group of

scientists and medical microbiologists in contact. Dissemination

of information has been achieved through ASM, NRL, RCPA QAP,

various direct emails and the national email routing ‘SERSIG’,

which links all members with requests for information or help in

resolving problems. Now SERSIG has 419 members.

At the ASM annual scientific meeting each year, we organise a

workshop with topics including QAP, case studies and issues with

diagnostic kit failures or problems. Previous workshops connected

with ASM annualmeetings in Adelaide,Melbourne and Sydneywere

organised by Ros Escott (RCPA QAP now retired). In 2014 a molec-

ular diagnosis workshop was held on the Saturday afternoon at the

Peter Doherty Institute prior to the national meeting. The meeting

was chaired by Wayne Dimech and Darren Jardine with over 80

attending. It included topics on molecular testing, validation and

standards, choosing equipment and new technologies.

An Annual General Meeting of the SIG is organised by the convenor

at each yearlymeeting and it includes branch reports, industry input

anddiscussionof current topics. InMelbourne last year 17members

attended, reports were tabled for NSW, QLD, VIC, SA andWA, and a

call for nominations for new national convenor, secretary and

workshop organiser was made.

After over 10 years of service as national convenor and serving as

secretary/treasurer of the NSW branch since 1993, I will be handing

over the duties and responsibilities of the ASM CS&M SIG to the

new convenor. Coordinating, participating and helping with all the

events and discussions of this SIG has been a pleasure for over

22 years. I would like to thank Wayne for his help and advice as

secretary and administrator of the SERSIG email routing and espe-

cially thank Sheena for being the driving force behind the working

group, organising teleconferences, emails to members and letters

to governing bodies. I trust the Clinical Serology and Molecular

SIG will continue to meet the needs of members by providing

useful and timely scientific information about diagnostic serology

and molecular techniques and related matters. I am sure the SIG

will be left in good hands with the help of the new executive.

New office bearers for the SIG are:

Convenor: Linda Hueston, Pathology West – ICPMR, Westmead,

NSW.


Secretary: Megan Wieringa, Monash Health, Clayton, Victoria.


Workshoporganiser:BruceWong,PaLMS,RNSH,St Leonards,NSW.


State Branch Convenors 2014

NSW: Deane Byers, RCPA.


Victoria: Wayne Dimech (retiring), NRL.


Queensland: Cheryl Bletchley and Theo Sloots (retiring).


South Australia: Trish Hahesy, IMVS.


Western Australia: Justin Morgan, PathWest Laboratory

Medicine WA.


Tasmania: Louise Cooley, RHH.


ASMAffairs


Report from the ASM Antimicrobial SpecialInterest Group (ASIG)

John Merlino, on behalfof ASIG

ASM ASIG Convenor/ChairEmail:[email protected]

The ASM Antimicrobial Special Interest Group (ASIG) is made up

of colleagues and ASM members you have an interest in antimicro-

bials. Members of the ASIG committee include medical, veterinary,

CDS Users, mycology and parasitology consultants. The Antimicro-

bial SIG is one of the largest Special Interest Groups in the ASM.

In 2012 we had over 923 ASIG members. In 2013 we had 721

members listed under ASIG. Some of our members have retired.

Numbers are yet to be finalised in 2014.

In 2014 ASM ASIG members were actively involved with local and

national ASM Branch meetings in various states – this is usual in the

form of communication and newsletters. Committee actively com-

municates via emails and website www.asig.org.au on emerging

issues – methodology on susceptibility testing and standards on

antimicrobials and antimicrobial resistance. Members of the com-

mittee actively take part in planning workshops titles and speakers,

e.g. Annual Scientific ASM Meetings – this has been the focus for

many years. Discussions on antimicrobial issues take place at these

meetings.

In Melbourne in 2014, the Antimicrobial Special Interest Group

Workshop on Antimicrobial Resistance focused onMALDI-TOF and

Susceptibility Testing with both CLSI and EUCAST methodology.

This was a DRY Workshop with practical slide demonstrations with

discussions and a QUIZ with equipment such as buzzers provide

via the RCPA. Speakers’ presentations are available on (powerpoint

in PDF) on the ASIG website www.asig.org.au, http://asig.org.

au/australian-society-for-microbiology-asm-2014-annual-scientific-

meeting-melbourne-australia-workshop-presentations/.

TheBioMerieux Identifying Resistance Award in 2014went 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-

therapy (ISC) after a formal invitation. Some of our members who

specialise in specific antimicrobial areashavebeen invited and taken

part in symposiums andother events at their own costs.Members of

ASIG committee actively communicate with other local and inter-

national antimicrobial groups on emerging issues in antimicrobial

resistance for example with the MRSA International Committee.

This remains ongoing in 2014–15. ASIG Members have also been

involved in EuropeanCongress Clinical Microbiology and Infectious

Diseases (ECCMID)meetings overseas – expensesmet by their own

trust fund not ASM. Members have been invited and involved in

writing articles on antimicrobials for Microbiology Australia and

overseas journals. When requested ASIG members who specialise

in specific areas of antimicrobials interests review articles for local

and international journals.

The ASIG convenor/committee and members communicates with

Division1Chairs, informally invitingoverseas speakers inorganising

symposiums and workshops at ASM Annual Scientific Meetings.

ASIG continues to review submissions and comments from other

Medical andAntimicrobialGroups in regards to antimicrobial issues,

e.g. Royal College of Pathologists Australasia Advisory Committee,

AIMS, NATA, NPAAC, with the ASM Clinical Standing Committee

when required. ASIG continues support of teaching and research

activities where possible by seeking speakers for events. ASIG

promotes theadvancementofASMSIGantimicrobials bypresenting

lectures/visits to Universities and Colleges nationally and interna-

tionally as required.

ASIG is involved with the Continuing Education Program, involved

with the AIMS APACE Official Certificate of Attendance is given out

at all ASIG workshops at National meetings. These certificates are

important for professional developments and career advancements

in industry. As the convenor I thank all our members and sponsors

for their continued support.

ASMAffairs


Annual Scientific Meeting and Trade Exhibition

[email protected]://asmmeeting.theasm.org.au/

12-15 July 2015QT CANBERRA1 London Circuit, Canberra

Rubbo Oration Janet Jansson, Pacific Northwest National Laboratory, USABazeley Oration Yoshihiro Kawaoka, University Wisconsin -Madison, USA, University Tokyo, JapanPlenary 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.

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