Science & Society

Managing AmphibianDisease with SkinMicrobiotaDouglas C. Woodhams,1,*Molly Bletz,2

Jordan Kueneman,3 andValerie McKenzie3

The contribution of emergingamphibian diseases to the sixthmass extinction is driving innovativewildlife management strategies,including the use of probiotics. Bio-augmentation of the skin muco-some, a dynamic environmentincluding host and microbial com-ponents, may not provide a general-ized solution. Multi-omicstechnologies and ecological contextunderlie effective implementation.

Global population declines and extinctionsof over 200 amphibian species have beenlinked to the fungus Batrachochytriumdendrobatidis (Bd). The hydrophilic chytridfungus infects amphibian skin, causing thedisease chytridiomycosis. Recent workputs forth the hypothesis that a hyperviru-lent Global Pandemic Lineage of Bd (Bd-GPL) may have emerged, in part, as aresult of hybridization among disparateBd genotypes brought into contact byhuman movement of amphibians [1],and that this lineage is associated withglobal amphibian declines. A newlydescribed salamander chytrid, Batracho-chytrium salamandrivorans (Bsal), is caus-ing recent declines in Europe andthreatens both other European salaman-der populations and the highly diverseNorth American salamanders. Evidenceagain points toward human-facilitatedtranscontinental movement of animals inspreading Bsal to a new geographicregion of susceptible amphibians [2]. Inaddition to proposed trade restrictions

and containment of Bsal, developmentof new tools for disease management isurgently needed.

There are a variety of disease manage-ment strategies, including removal of dis-ease reservoirs (e.g., Bd-tolerant,invasive amphibians), habitat alterationby reducing canopy cover to increasebasking opportunities, management ofzoospore micro-predators, and fungicideapplications. However, many of thesestrategies are either not feasible or arenot effectual in amphibian wetland habi-tats, particularly in remote and pristineregions. A relatively new strategy har-nesses the natural enemies of fungalpathogens, which reside right on theamphibians’ skin as symbiotic micro-biota, that are capable of inhibiting path-ogen colonization and establishment.Thus, we consider this approach to bea novel application of probiotics or bene-ficial bacteria that provide pathogendefense for the amphibian host [3]. Wehave recently produced a database ofsuch beneficial bacteria with a corre-sponding culture collection maintainedat UMass Boston and the Culture Collec-tion of Switzerland [4]. Most of these bac-teria were tested in co-culture assaysagainst Bd, and some have been usedsuccessfully to promote disease resis-tance when applied to frog and salaman-der skin [3].

The mechanisms by which bacteria (andother symbionts, including viruses, fungi,and protozoans) are able to promote dis-ease resistance are not well known. Somebacteria are able to promote host immunedefenses, and others compete with invad-ing pathogens directly. Direct competitionamong microbes often leads to secretedtoxins, such as small antibiotic molecules.In several cases, Bd inhibition was asso-ciated with the production of bacterialsecondary metabolites. For example,Pseudomonas fluorescens produces thecompound 2,4-diacetylphloroglucinol(DAPG) and Janthinobacterium lividumproduces indole-3-carboxaldehyde (I3C)

and violacein, all of which can inhibit Bdgrowth [3].

The production of bacteriocins and volatileorganic compounds (VOCs), while lessexplored, may also be responsible forbacterial inhibition of Bd. Bacteriocinsare a group of antimicrobial peptides pro-duced by bacteria and archaea. In humansystems, bacteriocins produced by resi-dent bacteria are known to have a bacte-rial pathogen-inhibiting function [5], andsome bacteriocins are antifungal. Thesame could be true for amphibians’ bac-terial symbionts; however, this remainsunexplored. In addition to the use of thebacteriocin-producing bacteria as probi-otics for amphibians, purified bacteriocins(e.g., pheromonicins [6]) could also beused to treat Bd infection, especially incaptive situations, and may be safer thantraditional antifungal compounds foramphibians. Currently, itraconazole isthe best antifungal treatment option foramphibians when used correctly. Antifun-gal treatments include potential sideeffects and negative impacts on non-targetmicrobiota.

In addition to antibiotic production andantifungal activity when in co-culture [4],some bacteria appear to have long-dis-tance effects facilitated by VOCs. Here weshow that bacteria confined to one side ofa split-Petri plate are able to inhibit Bdgrowth on the other side of the plate,without physical contact (Figure 1). Thus,we suspect that aerosolized VOCs inhibitfungal growth. Indeed, the botany litera-ture has many examples of soil bacteriathat are able to promote plant health andlimit fungal disease by VOC production.For example, some Pseudomonas bacte-ria produce compounds, including phlor-oglucinols and hydrogen cyanide, thatsuppress plant disease [7]. Multiple soilbacteria with VOC-mediated antifungalactivity [8] have been found on amphib-ians’ skin. Similarly, researchers studyingPseudogymnoascus destructans, the eti-ological agent of white-nose syndromein bats, have recently proposed using

Trends in Microbiology, March 2016, Vol. 24, No. 3 161

Experimental isolate Representa�ve image ofBd (10x, 400 µm scale bar)

Mean % Bd area (SD) T (df = 4) P Cyantesmo test strip

(blue indicates HCN)

Janthinobacterium lividum 9.5 (3.4) 8.439 0.001

Pseudomonas sp. 9.0 (2.5) 8.87 0.001

Rhodococcus fascians 0.0 (0.0) 11.23 <0.0001

Control 52.0 (8.0)

Figure 1. Amphibian Skin Bacteria Producing Volatile Antifungal Compounds. Preliminary data showthat amphibian skin bacteria produce aerosol volatile compounds capable of inhibiting growth of the fungalpathogen Batrachochytrium dendrobatidis (Bd). Top: Split-Petri dishes (VWR) show a bacterium on the bottomhalf of the left plate isolated from a southern leopard frog, Lithobates sphenocephala, (Rhodococcus fascians –

matching GenBank accession number HG796188) and no growth of Bd (top half of plate), and control (rightplate) with abundant Bd growth. Note that secondary metabolites from R. fascians collected in liquid media didnot inhibit Bd. The antifungal volatile organic compounds (VOCs) released from the bacteria are underinvestigation and may lead to disease-management strategies to improve amphibian conservation. Hydrogencyanide (HCN) production was not indicated based on Cyantesmo strip tests (Macherey-Nagel GmbH & Co.),but was indicated in a similarly tested Pseudomonas isolate from Colombia (VF35). Bottom: We quantified thearea of Bd growth in split-Petri dish experiments with VOC-producing antifungal isolates from amphibian skincompared to control growth (experimental design adapted from [8], artificially colored red area calculated withImageJ, statistics with IBM SPSS Statistics v.23). R. fascians completely inhibited Bd. Inhibition of Bd by isolatesproducing VOCs is included in updates to the Antifungal Isolates Database (4).

VOC-producing soil bacteria (Rhodococ-cus) in hibernation caves to kill spores andreduce the fungal pathogen [9]. In vivostudies are needed to examine live

162 Trends in Microbiology, March 2016, Vol. 24, No. 3

amphibians for the production of antifun-gal compounds identified in cultures, andto test treatment of infected amphibians.Perhaps VOC-producing bacteria can be

deployed at amphibian-rich habitats (i.e.,breeding sites) to reduce the zoosporepool, thereby facilitating population per-sistence. Instead of the stench of disease,recollecting miasma theory, perhaps hostodors can signal the presence of antifun-gal microbial defenses and contribute tobehavioral ecology and sexual selectionof resistant hosts. In other words, frogsmay be able to smell VOC-producingmicrobiota, and if odor is linked withdisease resistance, protected partnersmay have a selective advantage in matechoice.

The goal of microbial therapy is to increasesurvival of amphibians through augment-ing skin microbes that inhibit pathogensdirectly or indirectly by promoting hostimmunity. Probiotic bioaugmentationmay lead to herd immunity in amphibianpopulations or communities [3]. The eco-logical context and biotic resistance toprobiotics – climate, host immuneresponse, and microbial community com-position or diversity – will shape theireffects [10]. A primary criterion for aneffective probiotic is the ability to colonizeand persist on the amphibian's skin at anabundance high enough to achieve hostprotection [3]. For some species this hasbeen a major obstacle, such as the extinct(in the wild) Panamanian golden frog, Ate-lopus zeteki [11] or the Panamanian rocketfrog, Colostethus panamansis [12]. In anumber of other systems, microbial com-munities become established early indevelopment, even in the human womb[13]. Thus, probiotics applied early indevelopment may colonize open nichesand establish in concert with host immu-nity. We found that some amphibian skin-associated bacteria can persist throughlife-history transitions from aquatic larvaethrough terrestrial adult stages (Figure 2,data from [14]). Identification of core anti-fungal community members, and aug-mentation of these members early indevelopment, may prove effective. Anantifungal Chryseobacterium sp. wasone core member found across life stages.While not a core member, J. lividum has

Tadpole Metamorph

27 5 9

18

43

17

3

Juvenile + Adult

n core OTUs Phylum1

1

22

11

AcidobacteriaAc�nobacteriaBacteriodetesProteobacteriaVerrucomicrobia

Figure 2. Prevalent Skin Bacteria Persisting through Life-history. The bacteria found on the skin ofboreal toad, Anaxyrus boreas, aquatic tadpoles, recent metamorphs, and terrestrial juveniles and adultsdemonstrate a shifting community with a significant overlap in membership. The Venn diagram includesnumbers of operational taxonomic units (OTUs) found in �90% of individuals in each life-history stage (n =8 tadpoles, 8 metamophs, and 16 juveniles/adults) field-sampled concurrently at Trout creek, Colorado. Forexample, in the tadpole lifestage, there are 27 core bacterial OTUs that are only found on that lifestage, 5 coreOTUs are found on both tadpoles and metamorphs, and 18 core OTUs are found on both tadpoles and adults.Seventeen core bacterial OTUs are found across all boreal toad life stages including a Chryseobacterium sp.identified in culture with anti-Bd metabolite production. Details of the study are found in [14], and indicatepersistant potential probiotics closely associated with the host. Photos from D. Brewbooks and J. N. Stuart.

violacein-producing antifungal strains thatcan reduce mortality from chytridiomyco-sis, and is occasionally present on tad-poles. Thus, augmentation of J. lividumat the tadpole stage by probiotics or pre-biotics (nutrients favoring beneficial bacte-ria) may prove effective. Furthermore, inone study, Escherichia coli was engineeredto produce violacein [15], and commonamphibian skin bacteria could be similarlymanipulated, leading to more consistentprobiotic establishment on hosts. Bioticresistance theory may not apply perfectlyto microbiota in understanding colonizationresistance because therapies avoid intro-ducing novel organisms into naïve popula-tions and there are both host(environmental) and microbial (competition)factors. That said, microbial diversity and

composition may have an understudiedinfluence on probiotic effectiveness.

The current focus on individual bacteriaas probiotics to mitigate chytridiomyco-sis is likely over-simplistic. The use ofmultispecies probiotics or whole-com-munity transplants [12] are avenues forfuture exploration. Additionally, multi-omics approaches that integrate func-tional aspects of interacting microbialcommunities [14], metagenomic descrip-tions of the microbial communities, andmetabolomic descriptions of the activemucosal compounds have great poten-tial to advance this field. For example,network analyses used by Kuenemanet al. [14] show that some bacterialgroups, including the Burkholderiales

and Pseudomonadales, negatively co-occur with fungi. These correlationalanalyses, along with culture and in vivostudies incorporating the ecological con-text of the mucosome, help focus probi-otic development. One frontier is theidentification of bacterial genes associ-ated with antifungal function. Antifungalgenes are unlikely to be expressed con-sistently under common culture condi-tions, and isolate identification by 16SrRNA gene sequencing is not necessarilya reliable indication of the antifungalcapacity. Whole genome sequencingmay thus lead to better diagnosticsof antifungal function of microbialcommunities.

In addition to developing conservationstrategies, future studies may also bedirected toward testing ecological theory.For example, to what extent are hosts andmicrobiota co-evolved? Is the microbiotaheritable? To what extent are recoveringamphibian populations adapting to Bd bychanges in microbiota or microbial-immune interactions? Are there ecologicaltrade-offs between a microbiota-baseddefense and a host immune systemdefense? Can microbial VOCs signal theimmune condition of hosts, or affectbehavior of conspecific hosts?

In addition to these research questions, acommon bioethical concern of conserva-tion biologists is: What are the ecologicalconsequences of probiotic applications,particularly in sensitive habitats with otherendangered wildlife species? This con-cern is balanced by the consequence ofno action and continuing populationlosses. Current standards for ecologicaluse of bioaugmentation strategies include:(i) the use of native microbes only, perhapscultured from the skin of an amphibian co-existing with Bd. The host may either tol-erate low-level infection, or resist infection[3]. (ii) Tailored management within sys-tems should also examine non-targeteffects on aquatic and soil microbes,and the microbiota of non-target hosts.Furthermore, there are many potential

Trends in Microbiology, March 2016, Vol. 24, No. 3 163

applications of probiotic approaches toamphibians in aquaculture systems andthe pet-trade that could have a significantimpact toward reducing the spread offungal pathogens via these trade routes.Novel tools that target the pathogen butlimit toxicity to the host, such as probiot-ics, are sorely needed in these sectors.While there is much to learn in the field ofwildlife microbial ecology, probiotics holdgreat potential for directed disease man-agement and conservation intervention.

AcknowledgmentsWe thank Brandon LaBumbard and Kate Dutton-

Regester for assistance with VOC experiments, Kevin

Minbiole and Eric Baitchman for helpful discussion,

and Vicky Flechas, Whitney Holden and Louise Roll-

ins-Smith for bacterial cultures. DCW was supported

by the UMass Boston Endowed Faculty Career Devel-

opment Award, and VJM was supported by U.S. NSF

grant (DEB:1146284).

1Department of Biology, University of Massachusetts

Boston, Boston, MA, USA2Zoological Institute, Technische Universität

Braunschweig, Braunschweig, Germany3Department of Ecology and Evolutionary Biology,

University of Colorado, Boulder, CO, USA

*Correspondence: dwoodhams@gmail.com

(D.C. Woodhams).

http://dx.doi.org/10.1016/j.tim.2015.12.010

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SpotlightHIV-1 EnvelopeUnder AttackWei Wei1,2 andXiao-Fang Yu1,2,*

The human immunodeficiencyvirus type 1 (HIV-1) envelope(Env) plays a critical role in viralreplication and represents a poten-tial target for host antiviral factors.Recent work by Tada and col-leagues identifies membrane-associated-RING-CH8 (MARCH8)as a potent anti-HIV factor blockingvirion incorporation of Env. Thus,MARCH8 joins a growing list ofhost factors attacking HIV-1 Env.

The HIV-1 Env glycoprotein plays criticalroles in viral replication, tropism, and path-ogenesis. Env is first synthesized as apolyprotein, gp160, and is subsequentlycleaved by cellular proteases in the Golgiapparatus into gp120 and gp41. Theproper synthesis, modification, folding,cleavage, cell surface targeting, and virionincorporation of Env are all essential stepsin the generation of infectious HIV-1 par-ticles. Consequently, these steps are allvulnerable targets for attack by hostdefense factors (Figure 1).

A recent study by Tada and colleagues,published in Nature Medicine, has illus-trated the importance of this concept ofhost defense against HIV-1 Env [1]. Theseauthors identify MARCH8 as a novel anti-viral factor suppressing HIV-1 Env surfaceexpression and virion incorporation. Likemany scientific discoveries, their findingcame from an unexpected observation:that the transduction efficiency oflentiviruses produced from MARCH8-expressing cells was 65-fold lower thanthat of control lentiviruses, suggesting anantiviral role for this protein. Tada andcolleagues demonstrated, in a logicalseries of experiments, that MARCH8 isa novel antiviral factor against diverseviruses in virus-producing cells. MARCH8expression in target cells showed no effecton viral infectivity, indicating that its antiviralactivity is the result of its expression in theproducer cells. Also, MARCH8 expressionin producer cells did not influence the pro-duction of HIV-1 virions, as monitored byviral Gag proteins. The blockage in theinfection of the viruses produced fromMARCH8-expressing cells occurred priorto reverse transcription and integration,and in fact was due to a marked defectin viral entry. Finally, detailed analysis ofreleased viral particles revealed that thelevel of Env proteins on HIV-1 virions pro-duced from MARCH8-expressing cellswas dramatically decreased.

MARCH8 is a member of the MARCHfamily of RING-finger E3 ubiquitin ligasesand is mainly found in endosomes, lyso-somes, and vacuoles, but also on theplasma membrane. MARCH8 inducesthe ubiquitylation of substrate proteinsand regulates their vesicular transportbetween membrane compartments.Cell-surface expression of several cellularglycoproteins is specifically downregu-lated by MARCH8 [2]. The anti-HIV-1activity of MARCH8 is dependent on itsE3 ubiquitin ligase activity. As a potentialviral restriction factor, MARCH8 is highlyexpressed in monocyte-derived macro-phages (MDM) and dendritic cells(MDDC). The expression of endogenous