Advertisement

Functional Redundancy of Batrachochytrium dendrobatidis Inhibition in Bacterial Communities Isolated from Lithobates clamitans Skin

Abstract

The cutaneous microbial community can influence the health of amphibians exposed to Batrachochytrium dendrobatidis (Bd), a fungal pathogen that has contributed to recent amphibian declines. Resistance to Bd in amphibian populations is correlated with the presence of anti-Bd cutaneous microbes, which confer disease resistance by inhibiting Bd growth. I aimed to determine if green frogs (Lithobates clamitans), an abundant and widely distributed species in New Jersey, harbored bacteria that inhibit Bd and whether the presence and identity of these microbes varied among sites. I used in vitro challenge assays to determine if bacteria isolated from green frog skin could inhibit or enhance the growth of Bd. I found that green frogs at all sites harbored anti-Bd bacteria. However, there were differences in Bd inhibition capabilities among bacterial isolates identified as the same operational taxonomic unit (OTU), lending support to the idea that phylogenetic relatedness does not always predict Bd inhibition status. Additionally, anti-Bd bacterial richness did not vary by site, but the composition of anti-Bd bacterial taxa was distinct at each site. This suggests that there is functional redundancy of Bd inhibition across unique communities of anti-Bd symbionts found on frogs at different sites. These findings highlight the need to better elucidate the structure-function relationship of microbiomes and their role in disease resistance.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. 1.

    Pflughoeft KJ, Versalovic J (2012) Human microbiome in health and disease. Annu. Rev. Pathol. 7:99–122. https://doi.org/10.1146/annurev-pathol-011811-132421

  2. 2.

    Cho I, Blaser MJ (2012) The human microbiome: at the interface of health and disease. Nat Rev Genet 13:260–270. https://doi.org/10.1038/nrg3182

  3. 3.

    Grice EA, Segre JA (2011) The skin microbiome. Nat Rev Microbiol 9:244–253. https://doi.org/10.1038/nrmicro2537

  4. 4.

    Hoyt JR, Cheng TL, Langwig KE, Hee MM, Frick WF, Kilpatrick AM (2015) Bacteria isolated from bats inhibit the growth of Pseudogymnoascus destructans, the causative agent of white-nose syndrome. PLoS One 10:e0121329. https://doi.org/10.1371/journal.pone.0121329

  5. 5.

    Walke JB, Belden LK (2016) Harnessing the microbiome to prevent fungal infections: lessons from amphibians. PLoS Pathog. 12:e1005796. https://doi.org/10.1371/journal.ppat.1005796

  6. 6.

    Bletz M, Kelly M, Sabino-Pinto J et al (2018) Disruption of skin microbiota contributes to salamander disease. Proc. R. Soc. B Biol. Sci. 285:20180758. https://doi.org/10.1098/rspb.2018.0758

  7. 7.

    Becker MH, Harris RN (2010) Cutaneous bacteria of the redback salamander prevent morbidity associated with a lethal disease. PLoS One 5:e10957. https://doi.org/10.1371/journal.pone.0010957

  8. 8.

    Woodhams DC, Vredenburg VT, Simon M-A, Billheimer D, Shakhtour B, Shyr Y, Briggs CJ, Rollins-Smith LA, Harris RN (2007) Symbiotic bacteria contribute to innate immune defenses of the threatened mountain yellow-legged frog, Rana muscosa. Biol. Conserv. 138:390–398. https://doi.org/10.1016/j.biocon.2007.05.004

  9. 9.

    Lam BA, Walke JB, Vredenburg VT, Harris RN (2010) Proportion of individuals with anti-Batrachochytrium dendrobatidis skin bacteria is associated with population persistence in the frog Rana muscosa. Biol. Conserv. 143:529–531. https://doi.org/10.1016/j.biocon.2009.11.015

  10. 10.

    Harris RN, Brucker RM, Walke JB, Becker MH, Schwantes CR, Flaherty DC, Lam BA, Woodhams DC, Briggs CJ, Vredenburg VT, Minbiole KPC (2009) Skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus. ISME J 3:818–824. https://doi.org/10.1038/ismej.2009.27

  11. 11.

    Bletz MC, Loudon AH, Becker MH, Bell SC, Woodhams DC, Minbiole KPC, Harris RN (2013) Mitigating amphibian chytridiomycosis with bioaugmentation: characteristics of effective probiotics and strategies for their selection and use. Ecol. Lett. 16:807–820. https://doi.org/10.1111/ele.12099

  12. 12.

    Belden LK, Harris RN (2007) Infectious diseases in wildlife: the community ecology context. Front. Ecol. Environ. 5:533–539. https://doi.org/10.1890/060122

  13. 13.

    Bright M, Bulgheresi S (2010) A complex journey: transmission of microbial symbionts. Nat Rev Microbiol 8:218–230. https://doi.org/10.1038/nrmicro2262

  14. 14.

    Muletz CR, Myers JM, Domangue RJ, Herrick JB, Harris RN (2012) Soil bioaugmentation with amphibian cutaneous bacteria protects amphibian hosts from infection by Batrachochytrium dendrobatidis. Biol. Conserv. 152:119–126. https://doi.org/10.1016/j.biocon.2012.03.022

  15. 15.

    McKenzie VJ, Bowers RM, Fierer N et al (2012) Co-habiting amphibian species harbor unique skin bacterial communities in wild populations. ISME J 6:588–596. https://doi.org/10.1038/ismej.2011.129

  16. 16.

    Kueneman JG, Parfrey LW, Woodhams DC, Archer HM, Knight R, McKenzie VJ (2014) The amphibian skin-associated microbiome across species, space and life history stages. Mol. Ecol. 23:1238–1250. https://doi.org/10.1111/mec.12510

  17. 17.

    Fitzpatrick BM, Allison AL (2014) Similarity and differentiation between bacteria associated with skin of salamanders (Plethodon jordani) and free-living assemblages. FEMS Microbiol. Ecol. 88:482–494. https://doi.org/10.1111/1574-6941.12314

  18. 18.

    Walke JB, Becker MH, Loftus SC, House LL, Cormier G, Jensen RV, Belden LK (2014) Amphibian skin may select for rare environmental microbes. ISME J 8:2207–2217. https://doi.org/10.1038/ismej.2014.77

  19. 19.

    Griffiths SM, Harrison XA, Weldon C, Wood MD, Pretorius A, Hopkins K, Fox G, Preziosi RF, Antwis RE (2018) Genetic variability and ontogeny predict microbiome structure in a disease-challenged montane amphibian. ISME J 1:2506–2517. https://doi.org/10.1038/s41396-018-0167-0

  20. 20.

    Franzenburg S, Walter J, Kunzel S, Wang J, Baines JF, Bosch TCG, Fraune S (2013) Distinct antimicrobial peptide expression determines host species-specific bacterial associations. Proc. Natl. Acad. Sci. 110:E3730–E3738. https://doi.org/10.1073/pnas.1304960110

  21. 21.

    Goodrich JK, Waters JL, Poole AC, Sutter JL, Koren O, Blekhman R, Beaumont M, van Treuren W, Knight R, Bell JT, Spector TD, Clark AG, Ley RE (2014) Human genetics shape the gut microbiome. Cell 159:789–799. https://doi.org/10.1016/j.cell.2014.09.053

  22. 22.

    Walke JB, Becker MH, Loftus SC, House LL, Teotonio TL, Minbiole KPC, Belden LK (2015) Community structure and function of amphibian skin microbes: an experiment with bullfrogs exposed to a chytrid fungus. PLoS One 10:e0139848. https://doi.org/10.1371/journal.pone.0139848

  23. 23.

    Bates KA, Clare FC, O’Hanlon S, Bosch J, Brookes L, Hopkins K, McLaughlin EJ, Daniel O, Garner TWJ, Fisher MC, Harrison XA (2018) Amphibian chytridiomycosis outbreak dynamics are linked with host skin bacterial community structure. Nat. Commun. 9:1–11. https://doi.org/10.1038/s41467-018-02967-w

  24. 24.

    Rebollar EA, Hughey MC, Medina D, Harris RN, Ibáñez R, Belden LK (2016) Skin bacterial diversity of Panamanian frogs is associated with host susceptibility and presence of Batrachochytrium dendrobatidis. ISME J 10:1682–1695. https://doi.org/10.1038/ismej.2015.234

  25. 25.

    Belden LK, Hughey MC, Rebollar EA, Umile TP, Loftus SC, Burzynski EA, Minbiole KPC, House LL, Jensen RV, Becker MH, Walke JB, Medina D, Ibáñez R, Harris RN (2015) Panamanian frog species host unique skin bacterial communities. Front. Microbiol. 6. https://doi.org/10.3389/fmicb.2015.01171

  26. 26.

    Harris RN, James TY, Lauer A, Simon MA, Patel A (2006) Amphibian pathogen Batrachochytrium dendrobatidis is inhibited by the cutaneous bacteria of amphibian species. Ecohealth 3:53–56. https://doi.org/10.1007/s10393-005-0009-1

  27. 27.

    Bell SC, Alford RA, Garland S, Padilla G, Thomas AD (2013) Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay. Dis. Aquat. Org. 103:77–85. https://doi.org/10.3354/dao02560

  28. 28.

    Antwis RE, Preziosi RF, Harrison XA, Garner TWJ (2015) Amphibian symbiotic bacteria do not show universal ability to inhibit growth of the global pandemic lineage of Batrachochytrium dendrobatidis. Appl. Environ. Microbiol. 81:AEM.00010-15. https://doi.org/10.1128/AEM.00010-15

  29. 29.

    Becker MH, Walke JB, Murrill L, Woodhams DC, Reinert LK, Rollins-Smith LA, Burzynski EA, Umile TP, Minbiole KPC, Belden LK (2015) Phylogenetic distribution of symbiotic bacteria from Panamanian amphibians that inhibit growth of the lethal fungal pathogen Batrachochytrium dendrobatidis. Mol. Ecol. 24:1628–1641. https://doi.org/10.1111/mec.13135

  30. 30.

    Woodhams DC, Alford RA, Antwis RE, Archer H, Becker MH, Belden LK, Bell SC, Bletz M, Daskin JH, Davis LR, Flechas SV, Lauer A, Gonzalez A, Harris RN, Holden WM, Hughey MC, Ibáñez R, Knight R, Kueneman J, Rabemananjara F, Reinert LK, Rollins-Smith LA, Roman-Rodriguez F, Shaw SD, Walke JB, McKenzie V (2015) Antifungal isolates database of amphibian skin-associated bacteria and function against emerging fungal pathogens. Ecology 96:595–595. https://doi.org/10.1890/14-1837.1

  31. 31.

    Kueneman JG, Woodhams DC, Harris R, Archer HM, Knight R, McKenzie VJ (2016) Probiotic treatment restores protection against lethal fungal infection lost during amphibian captivity. Proc. R. Soc. B 283:20161553. https://doi.org/10.1098/rspb.2016.1553

  32. 32.

    Kueneman JG, Woodhams DC, Van Treuren W et al (2016) Inhibitory bacteria reduce fungi on early life stages of endangered Colorado boreal toads (Anaxyrus boreas). ISME J 10:934–944. https://doi.org/10.1038/ismej.2015.168

  33. 33.

    Bletz MC, Perl RGB, Bobowski B et al (2017) Amphibian skin microbiota exhibits temporal variation in community structure but stability of predicted Bd-inhibitory function. ISME J 11:1521–1534. https://doi.org/10.1038/ismej.2017.41

  34. 34.

    Bletz MC, Myers J, Woodhams DC, Rabemananjara FCE, Rakotonirina A, Weldon C, Edmonds D, Vences M, Harris RN (2017) Estimating herd immunity to amphibian chytridiomycosis in Madagascar based on the defensive function of amphibian skin bacteria. Front. Microbiol. 8:1751. https://doi.org/10.3389/FMICB.2017.01751

  35. 35.

    Bell SC, Garland S, Alford RA et al (2018) Increased numbers of culturable inhibitory bacterial taxa may mitigate the effects of Batrachochytrium dendrobatidis in Australian wet tropics frogs. Front. Microbiol. 9:1–14. https://doi.org/10.3389/fmicb.2018.01604

  36. 36.

    Antwis RE, Harrison XA (2018) Probiotic consortia are not uniformly effective against different amphibian chytrid pathogen isolates. Mol. Ecol. 27:577–589. https://doi.org/10.1111/mec.14456

  37. 37.

    Woodhams DC, Brandt H, Baumgartner S, Kielgast J, Küpfer E, Tobler U, Davis LR, Schmidt BR, Bel C, Hodel S, Knight R, McKenzie V (2014) Interacting symbionts and immunity in the amphibian skin mucosome predict disease risk and probiotic effectiveness. PLoS One 9:e96375. https://doi.org/10.1371/journal.pone.0096375

  38. 38.

    Gahl MK, Longcore JE, Houlahan JE (2012) Varying responses of northeastern North American amphibians to the chytrid pathogen Batrachochytrium dendrobatidis. Conserv. Biol. 26:135–141. https://doi.org/10.1111/j.1523-1739.2011.01801.x

  39. 39.

    Richards-Hrdlicka KL, Richardson JL, Mohabir L (2013) First survey for the amphibian chytrid fungus Batrachochytrium dendrobatidis in Connecticut (USA) finds widespread prevalence. Dis. Aquat. Org. 102:169–180. https://doi.org/10.3354/dao02552

  40. 40.

    Monsen-Collar K, Hazard L, Dussa R (2010) Comparison of PCR and RT-PCR in the first report of Batrachochytrium dendrobatidis in amphibians in New Jersey, USA. Herpetol Rev 41:460–462

  41. 41.

    Walke JB, Becker MH, Hughey MC, Swartwout MC, Jensen RV, Belden LK (2017) Dominance-function relationships in the amphibian skin microbiome. Environ. Microbiol. 19:3387–3397. https://doi.org/10.1111/1462-2920.13850

  42. 42.

    Daskin JH, Bell SC, Schwarzkopf L, Alford RA (2014) Cool temperatures reduce antifungal activity of symbiotic bacteria of threatened amphibians—implications for disease management and patterns of decline. PLoS One 9:e100378. https://doi.org/10.1371/journal.pone.0100378

  43. 43.

    Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Geneious. Bioinformatics. 28:1647–1649. https://doi.org/10.1093/bioinformatics/bts199

  44. 44.

    Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naïve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73:5261–5267. https://doi.org/10.1128/AEM.00062-07

  45. 45.

    R Core Team (2016) R: a language and environment for statistical computing. R Dev Core Team. doi: 3–900051–14-3

  46. 46.

    Fox J, Weisberg S, Adler D et al (2017) Companion to applied regression, Package ‘car’. Version 2.1-6. Available at: https://cran.r-project.org/web/packages/car/car.pdf. Accessed 2 Jan 2019

  47. 47.

    Oksanen J, Blanchet FG, Kindt R, et al. (2013) Package ‘vegan.’ R Packag ver 20–8. https://doi.org/10.4135/9781412971874.n145

  48. 48.

    Holden WM, Hanlon SM, Woodhams DC, Chappell TM, Wells HL, Glisson SM, McKenzie VJ, Knight R, Parris MJ, Rollins-Smith LA (2015) Skin bacteria provide early protection for newly metamorphosed southern leopard frogs (Rana sphenocephala) against the frog-killing fungus, Batrachochytrium dendrobatidis. Biol. Conserv. 187:91–102. https://doi.org/10.1016/j.biocon.2015.04.007

  49. 49.

    Muletz-Wolz CR, DiRenzo GV, Yarwood SA, Campbell Grant EH, Fleischer RC, Lips KR (2017) Antifungal bacteria on woodland salamander skin exhibit high taxonomic diversity and geographic variability. Appl. Environ. Microbiol. 83:1–13. https://doi.org/10.1128/AEM.00186-17

  50. 50.

    Hoshino T (2011) Violacein and related tryptophan metabolites produced by Chromobacterium violaceum: biosynthetic mechanism and pathway for construction of violacein core. Appl. Microbiol. Biotechnol. 91:1463–1475. https://doi.org/10.1007/s00253-011-3468-z

  51. 51.

    Becker MH, Brucker RM, Schwantes CR, Harris RN, Minbiole KPC (2009) The bacterially produced metabolite violacein is associated with survival of amphibians infected with a lethal fungus. Appl. Environ. Microbiol. 75:6635–6638. https://doi.org/10.1128/AEM.01294-09

  52. 52.

    Rebollar EA, Antwis RE, Becker MH, Belden LK, Bletz MC, Brucker RM, Harrison XA, Hughey MC, Kueneman JG, Loudon AH, McKenzie V, Medina D, Minbiole KPC, Rollins-Smith LA, Walke JB, Weiss S, Woodhams DC, Harris RN (2016) Using “omics” and integrated multi-omics approaches to guide probiotic selection to mitigate chytridiomycosis and other emerging infectious diseases. Front. Microbiol. 7:68. https://doi.org/10.3389/fmicb.2016.00068

  53. 53.

    Nemergut DR, Schmidt SK, Fukami T, O'Neill SP, Bilinski TM, Stanish LF, Knelman JE, Darcy JL, Lynch RC, Wickey P, Ferrenberg S (2013) Patterns and processes of microbial community assembly. Microbiol. Mol. Biol. Rev. 77:342–356. https://doi.org/10.1128/MMBR.00051-12

  54. 54.

    Louca S, Martiny AC, Polz MF et al (2018) Function and functional redundancy in microbial communities. Nat Ecol Evol 2:936–943

  55. 55.

    Loudon AH, Woodhams DC, Parfrey LW, Archer H, Knight R, McKenzie V, Harris RN (2014) Microbial community dynamics and effect of environmental microbial reservoirs on red-backed salamanders (Plethodon cinereus). ISME J 8:830–840. https://doi.org/10.1038/ismej.2013.200

  56. 56.

    Muletz Wolz CR, Yarwood SA, Campbell Grant EH, Fleischer RC, Lips KR (2017) Effects of host species and environment on the skin microbiome of Plethodontid salamanders. J. Anim. Ecol. 87:341–353. https://doi.org/10.1111/1365-2656.12726

  57. 57.

    Varela BJ, Lesbarrères DA, Ibáñez R, Green DM (2018) Environmental and host effects on skin bacterial community composition in Panamanian frogs. Front. Microbiol. 9:298. https://doi.org/10.3389/FMICB.2018.00298

  58. 58.

    Medina D, Hughey MC, Becker MH, Walke JB, Umile TP, Burzynski EA, Iannetta A, Minbiole KPC, Belden LK (2017) Variation in metabolite profiles of amphibian skin bacterial communities across elevations in the neotropics. Microb. Ecol. 74:227–238. https://doi.org/10.1007/s00248-017-0933-y

  59. 59.

    Longo AV, Savage AE, Hewson I, Zamudio KR (2015) Seasonal and ontogenetic variation of skin microbial communities and relationships to natural disease dynamics in declining amphibians. R. Soc. Open Sci. 2:140377. https://doi.org/10.1098/rsos.140377

  60. 60.

    Muletz-Wolz CR, Almario JG, Barnett SE, DiRenzo GV, Martel A, Pasmans F, Zamudio KR, Toledo LF, Lips KR (2017) Inhibition of fungal pathogens across genotypes and temperatures by amphibian skin bacteria. Front. Microbiol. 8:1551. https://doi.org/10.3389/fmicb.2017.01551

  61. 61.

    Woodhams DC, LaBumbard BC, Barnhart KL et al (2018) Prodigiosin, violacein, and volatile organic compounds produced by widespread cutaneous bacteria of amphibians can inhibit two Batrachochytrium fungal pathogens. Microb. Ecol. 75:1049–1062. https://doi.org/10.1007/s00248-017-1095-7

  62. 62.

    Scheuring I, Yu DW (2012) How to assemble a beneficial microbiome in three easy steps. Ecol. Lett. 15:1300–1307. https://doi.org/10.1111/j.1461-0248.2012.01853.x

  63. 63.

    Loudon AH, Holland JA, Umile TP, Burzynski EA, Minbiole KPC, Harris RN (2014) Interactions between amphibians’ symbiotic bacteria cause the production of emergent anti-fungal metabolites. Front. Microbiol. 5:441. https://doi.org/10.3389/fmicb.2014.00441

  64. 64.

    Vartoukian SR, Palmer RM, Wade WG (2010) Strategies for culture of “unculturable” bacteria. FEMS Microbiol. Lett. 309:1–7. https://doi.org/10.1111/j.1574-6968.2010.02000.x

  65. 65.

    Walke JB, Becker MH, Hughey MC, Swartwout MC, Jensen RV, Belden LK (2015) Most of the dominant members of amphibian skin bacterial communities can be readily cultured. Appl. Environ. Microbiol. 81:6589–6600. https://doi.org/10.1128/AEM.01486-15

  66. 66.

    Küng D, Bigler L, Davis LR, Gratwicke B, Griffith E, Woodhams DC (2014) Stability of microbiota facilitated by host immune regulation: informing probiotic strategies to manage amphibian disease. PLoS One 9:e87101. https://doi.org/10.1371/journal.pone.0087101

  67. 67.

    Robinson CJ, Bohannan BJM, Young VB (2010) From structure to function: the ecology of host-associated microbial communities. Microbiol. Mol. Biol. Rev. 74:453–476. https://doi.org/10.1128/MMBR.00014-10

  68. 68.

    Rebollar EA, Gutiérrez-Preciado A, Noecker C, Eng A, Hughey MC, Medina D, Walke JB, Borenstein E, Jensen RV, Belden LK, Harris RN (2018) The skin microbiome of the neotropical frog Craugastor fitzingeri: inferring potential bacterial-host-pathogen interactions from metagenomic data. Front. Microbiol. 9:1–12. https://doi.org/10.3389/fmicb.2018.00466

  69. 69.

    Medina D, Walke JB, Gajewski Z, Becker MH, Swartwout MC, Belden LK (2017) Culture media and individual hosts affect the recovery of culturable bacterial diversity from amphibian skin. Front. Microbiol. 8:1–14. https://doi.org/10.3389/fmicb.2017.01574

Download references

Acknowledgments

I thank Peter Morin, members of the Morin lab, Reid Harris, and anonymous reviewers for comments on the manuscript. I also thank Kevin Wyman and Paul Falkowski for use of the Falkowski lab’s spectrophotometer.

Funding

Funding for this work was provided by the New Jersey Water Resources Research Institute FY2016 Program, Project ID 2016NJ381B (USGS Grant Number G16AP00071) and a small grant award from the Rutgers University Ecology & Evolution Graduate Program.

Author information

Correspondence to Ariel Kruger.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kruger, A. Functional Redundancy of Batrachochytrium dendrobatidis Inhibition in Bacterial Communities Isolated from Lithobates clamitans Skin. Microb Ecol 79, 231–240 (2020). https://doi.org/10.1007/s00248-019-01387-7

Download citation

Keywords

  • Batrachochytrium dendrobatidis
  • Disease
  • Functional redundancy
  • Lithobates clamitans
  • Skin microbiome