Microbial Ecology

, Volume 73, Issue 2, pp 445–454 | Cite as

Characterization of the Cutaneous Bacterial Communities of Two Giant Salamander Subspecies

  • Obed Hernández-GómezEmail author
  • Steven J. A. Kimble
  • Jeffrey T. Briggler
  • Rod N. Williams
Host Microbe Interactions


Pathogens currently threaten the existence of many amphibian species. In efforts to combat global declines, researchers have characterized the amphibian cutaneous microbiome as a resource for disease management. Characterization of microbial communities has become useful in studying the links between organismal health and the host microbiome. Hellbender salamanders (Cryptobranchus alleganiensis) provide an ideal system to explore the cutaneous microbiome as this species requires extensive conservation management across its range. In addition, the Ozark hellbender subspecies (Cryptobranchus alleganiensis bishopi) exhibits chronic wounds hypothesized to be caused by bacterial infections, whereas the eastern hellbender (Cryptobranchus alleganiensis alleganiensis) does not. We assessed the cutaneous bacterial microbiome of both subspecies at two locations in the state of Missouri, USA. Through 16S rRNA gene-based amplicon sequencing, we detected more than 1000 distinct operational taxonomic units (OTUs) in the cutaneous and environmental bacterial microbiome. Phylogenetic and abundance-based dissimilarity matrices identified differences in the bacterial communities between the two subspecies, but only the abundance-based dissimilarity matrix identified differences between wounds and healthy skin on Ozark hellbenders. The higher abundance of OTUs on Ozark wounds suggests that commensal bacteria present on the skin and environment may be opportunistically colonizing the wounds. This brief exploration of the hellbender cutaneous bacterial microbiome provides foundational support for future studies seeking to understand the hellbender cutaneous bacterial microbiome and the role of the bacterial microbiota on chronic wounds of Ozark hellbenders.


Cryptobranchus alleganiensis Microbiome Wounds 



We would like to thank members of the Williams lab for assistance in revising this document. Special thanks also go to Jyothi Thimmapuram from the Purdue Bioinformatics Core, Phillip San Miguel and Paul Parker from the Purdue Genomics Core for assistance in project design, Bart Kraus for assistance in field collection, and Ardith Wang for assistance in sequencing preparation. Special thanks to the Missouri Department of Conservation for their interest and support of this project.

Compliance with Ethical Standards


Funding for this study was provided by Purdue University.

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Supplementary material

248_2016_859_MOESM1_ESM.docx (187 kb)
ESM 1 (DOCX 187 kb)


  1. 1.
    Federici E, Rossi R, Fidati L, Paracucchi R, Scargetta S, Montalbani E, Franzetti A, La Porta G, Fagotti A, Simonceli F, Cenci G, Di Rosa I (2015) Characterization of the skin microbiota in Italian stream frogs (Rana italica) infected and uninfected by a cutaneous parasitic disease. Microbes Environ 30:262–269CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    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 5:441CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Vredenburg VT, Briggs CJ, Harris RN (2011) Host-pathogen dynamics of amphibian chytridiomycosis: the role of the skin microbiome in health and disease. Fungal diseases: an emerging challenge to human, animal, and plant health. National Academic Press, Washington D.C., pp 342–355Google Scholar
  4. 4.
    Barker CJ, Gillett A, Polkinghorne A, Timms P (2013) Investigation of the koala (Phascolarctos cinereus) hindgut microbiome via 16S pyrosequencing. Vet Microbiol 167:554–564CrossRefPubMedGoogle Scholar
  5. 5.
    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 23:1238–1250CrossRefPubMedGoogle Scholar
  6. 6.
    Shabbir MZ, Park J, Muhammad K, Rabbani M, Rana MY, Harvill ET (2014) Culture independent analysis of respiratory microbiome of houbara bustard (Chlamydotis undulata) revealed organisms of public health significance. Int J Agric Biol 16:222–226Google Scholar
  7. 7.
    Ishak HD, Plowes R, Sen R, Kellner K, Meyer E, Estrada DA, Dowd SE, Mueller UG (2011) Bacterial diversity in Solenopsis invicta and Solenopsis geminata ant colonies characterized by 16S amplicon 454 pyrosequencing. Microb Ecol 61:821–831CrossRefPubMedGoogle Scholar
  8. 8.
    Daszak P, Cunningham AA, Hyatt AD (2003) Infectious disease and amphibian population declines. Divers Distrib 9:141–150CrossRefGoogle Scholar
  9. 9.
    Nickerson MA, Mays CE (1973) The hellbenders: North American ‘giant salamanders’. Milwaukee Public Museum, MilwaukeeGoogle Scholar
  10. 10.
    Foster RL, McMillan AM, Roblee KJ (2009) Population status of hellbender salamanders (Cryptobranchus alleganiensis) in the Allegheny River drainage of New York State. J Herpetol 43:579–588CrossRefGoogle Scholar
  11. 11.
    Hecht-Kardasz KA, Nickerson MA, Freake M, Colclough P (2012) Population structure of the hellbender (Cryptobranchus alleganiensis) in a Great Smoky Mountains stream. Bull Fla Museum Nat Hist 51:227Google Scholar
  12. 12.
    Briggler J, Utrup J, Davidson C, Humphries J, Groves J, Johnson T, Ettling J, Wanner M, Traylor-Holzer K, Reed D, Lindgren V, Byers O (2007) Hellbender population and habitat viability assessment: final report. IUCN/SSC Conservation Breeding Specialist Group, Apple ValleyGoogle Scholar
  13. 13.
    Burgmeier NG, Unger SD, Sutton TM, Williams RN (2011) Population status of the eastern hellbender (Cryptobranchus alleganiensis alleganiensis) in Indiana. J Herpetol 45:195–201CrossRefGoogle Scholar
  14. 14.
    Federal Register (2011) Endangered and threatened wildlife and plants; endangered status for the Ozark hellbender salamander. Fed Commun Comm 76:61956–61978Google Scholar
  15. 15.
    Wheeler BA, Prosen E, Mathis A, Wilkinson RF (2003) Population declines of a long-lived salamander: a 20+-year study of hellbenders, Cryptobranchus alleganiensis. Biol Conserv 109:151–156CrossRefGoogle Scholar
  16. 16.
    Hiler WR, Wheeler BA, Trauth SE (2005) Abnormalities in the Ozark hellbender (Cryptobranchus alleganiensis bishopi) in Arkansas: a comparison between two rivers with a historical perspective. J Arkansas Acad Sci 59:88–94Google Scholar
  17. 17.
    Nickerson CA, Ott CM, Castro SL, Garcia VM, Molina TC, Briggler JT, Pitt AL, Tavano JJ, Byram JK, Barrila J et al (2011) Evaluation of microorganisms cultured from injured and repressed tissue regeneration sites in endangered giant aquatic Ozark hellbender salamanders. PLoS One 6, e28906CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Wheeler BA, McCallum ML, Trauth SE (2002) Abnormalities in the Ozark hellbender, Cryptobranchus alleganiensis bishopi. J Arkansas Acad Sci 56:250–252Google Scholar
  19. 19.
    Irwin K (2008) Ozark hellbender long-term monitoring SWG project. Arkansas Game and Fish Commission, BentonGoogle Scholar
  20. 20.
    Hill GT, Mitkowski NA, Aldrich-Wolfe L, Emele LR, Jurkonie DD, Ficke A, Maldonado-Ramirez S, Lynch ST, Nelson EB (2000) Methods for assessing the composition and diversity of soil microbial communities. Appl Soil Ecol 15:25–36CrossRefGoogle Scholar
  21. 21.
    Pace NR (1997) A molecular view of microbial diversity and the biosphere. Science 276:734–740CrossRefPubMedGoogle Scholar
  22. 22.
    Jani AJ, Briggs CJ (2014) The pathogen Batrachochytrium dendrobatidis disturbs the frog skin microbiome during a natural epidemic and experimental infection. Proc Natl Acad Sci U S A 111:E5049–E5058CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Fitzpatrick BM, Allison AL (2014) Similarity and differentiation between bacteria associated with skin of salamanders (Plethodon jordani) and free-living assemblages. FEMS Microbiol Ecol 88:482–494CrossRefPubMedGoogle Scholar
  24. 24.
    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 8:830–840CrossRefPubMedGoogle Scholar
  25. 25.
    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, e96375CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ, Fierer N, Knight R (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci U S A 108:4516–4522CrossRefPubMedGoogle Scholar
  27. 27.
    Culp CE, Iii JOF, Belden LK (2007) Identification of the natubal bacterial microflora on the skin of eastern newts, bullfrog tadpoles and redback salamanders. Herpetologica 63:66–71CrossRefGoogle Scholar
  28. 28.
    Fierer N, Hamady M, Lauber CL, Knight R (2008) The influence of sex, handedness, and washing on the diversity of hand surface bacteria. Proc Natl Acad Sci U S A 105:17994–17999CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Bolger D, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for illumina sequence data. Bioinformatics 30:2114–2120CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI et al (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:2460–2461CrossRefPubMedGoogle Scholar
  32. 32.
    Bokulich NA, Subramanian S, Faith JJ, Gevers D, Gordon JI, Knight R, Mills DA, Caporaso JG (2013) Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat Methods 10:57–59CrossRefPubMedGoogle Scholar
  33. 33.
    DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72:5069–5072CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Caporaso JG, Bittinger K, Bushman FD, DeSantis TZ, Andersen GL, Knight R (2010) PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics 26:266–267CrossRefPubMedGoogle Scholar
  35. 35.
    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 73:5261–5267CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Price MN, Dehal PS, Arkin AP (2010) FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS One 5, e9490CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Chao A (1984) Nonparametric estimation of the number of classes in a population. Scand J Statist 11:265–270Google Scholar
  38. 38.
    Faith DP, Baker AM (2006) Phylogenetic diversity (PD) and biodiversity conservation: some bioinformatics challenges. Evol Bioinform Online 2:121–128Google Scholar
  39. 39.
    Shannon CE, Weaver W (1949) The mathematical theory of communication. University of Illinois Press, ChampaignGoogle Scholar
  40. 40.
    Bates D, Maechler M, Bolker BM, Walker SC (2015) Fitting linear mixed-effects models using lme4. J Stat Softw 67:1–48CrossRefGoogle Scholar
  41. 41.
    Lozupone C, Lladser ME, Knights D, Stombaugh J, Knight R (2011) UniFrac: an effective distance metric for microbial community comparison. ISME J 5:169–172CrossRefPubMedGoogle Scholar
  42. 42.
    Rees GN, Baldwin DS, Watson GO, Perryman S, Nielsen DL (2004) Ordination and significance testing of microbial community composition derived from terminal restriction fragment length polymorphisms: application of multivariate statistics. Anton Leeuw Int J G 86:339–347CrossRefGoogle Scholar
  43. 43.
    Okasen J, Blanchet FG, Friendly M, Kindt R, Legendre P, McGlinn D, Minchin PR, O’Hara RB, Simpson GL, Solymos P, Stevens MHH, Szoencs E, Wagner H (2016) Vegan: community ecology package. R package version 2.4-0Google Scholar
  44. 44.
    Hammer O, Harper DAT, Ryan PD (2001) PAST: paleontological statistics software package for education and data analysis. Palaeontol Electron 4:9Google Scholar
  45. 45.
    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. Roy Soc Open Sci 2:140377CrossRefGoogle Scholar
  46. 46.
    Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, Huttenhower C (2011) Metagenomic biomarker discovery and explanation. Genome Biol 12:R60CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, Brown CT, Porras-Alfaro A, Kuske CR, Tiedje JM (2014) Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res 42:D633–D642CrossRefPubMedGoogle Scholar
  48. 48.
    Atlas RM (2010) Handbook of microbiological media. CRC Press, Taylor and Francis Group, Boca RatonCrossRefGoogle Scholar
  49. 49.
    McKenzie VJ, Bowers RM, Fierer N, Knight R, Lauber CL (2012) Co-habiting amphibian species harbor unique skin bacterial communities in wild populations. ISME J 6:588–596CrossRefPubMedGoogle Scholar
  50. 50.
    Bataille A, Lee-Cruz L, Tripathi B, Kim H, Waldman B (2016) Microbiome variation across amphibian skin regions: implications for chytridiomycosis mitigation efforts. Microb Ecol 71:221–232CrossRefPubMedGoogle Scholar
  51. 51.
    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 8:2207–2217CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Crowhurst RS, Faries KM, Collantes J, Briggler JT, Koppelman JB, Eggert LS (2011) Genetic relationships of hellbenders in the Ozark highlands of Missouri and conservation implications for the Ozark subspecies (Cryptobranchus alleganiensis bishopi). Conserv Genet 12:637–646CrossRefGoogle Scholar
  53. 53.
    Feist SM, Briggler JT, Koppelman JB, Eggert LS (2014) Within-river gene flow in the hellbender (Cryptobranchus alleganiensis) and implications for restorative release. Conserv Genet 15:953–966CrossRefGoogle Scholar
  54. 54.
    Zilber-Rosenberg I, Rosenberg E (2008) Role of microorganisms in the evolution of animals and plants: the hologenome theory of evolution. FEMS Microbiol Rev 32:723–735CrossRefPubMedGoogle Scholar
  55. 55.
    Unger SD, Rhodes OE, Sutton TM, Williams RN (2013) Population genetics of the Eastern Hellbender (Cryptobranchus alleganiensis alleganiensis) across multiple spatial scales. PLoS One 8, e74180CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Olson ZH, Burgmeier NG, Zollner PA, Williams RN (2013) Survival estimates for adult Eastern Hellbenders and their utility for conservation. J Herpetol 47:71–74CrossRefGoogle Scholar
  57. 57.
    Bodinof CM, Briggler JT, Junge RE, Mong T, Beringer J, Wanner MD, Schuette CD, Ettling J, Millspaugh JJ (2012) Survival and body condition of captive-reared juvenile Ozark hellbenders (Cryptobranchus alleganiensis bishopi) following translocation to the wild. Copeia 2012:150–159CrossRefGoogle Scholar
  58. 58.
    Becker MH, Richards-Zawacki CL, Gratwicke B, Belden LK (2014) The effect of captivity on the cutaneous bacterial community of the critically endangered Panamanian golden frog (Atelopus zeteki). Biol Conserv 176:199–206CrossRefGoogle Scholar
  59. 59.
    Merrifield DL, Rodiles A (2015) The fish microbiome and its interactions with mucosal tissues. In: Mucosal health in aquaculture. Academic, Oxford, UK, pp 273–295CrossRefGoogle Scholar
  60. 60.
    Pfingsten RA (1989) The status and distribution of the hellbender, Cryptobranchus alleganiensis, in Ohio. Ohio J Sci 89:3Google Scholar
  61. 61.
    Lauer A, Simon MA, Banning JL, Lam BA, Harris RN (2008) Diversity of cutaneous bacteria with antifungal activity isolated from female four-toed salamanders. ISME J 2:145–157CrossRefPubMedGoogle Scholar
  62. 62.
    Michaels CJ, Antwis RE, Preziosi RF (2014) Impact of plant cover on fitness and behavioural traits of captive red-eyed tree frogs (Agalychnis callidryas). PLoS One 9(4):295207CrossRefGoogle Scholar
  63. 63.
    Roth T, Foley J, Worth J, Piovia-Scott J, Pope K, Lawler S (2013) Bacterial flora on Cascades frogs in the Klamath mountains of California. Comp Immunol Microb 36:591–598CrossRefGoogle Scholar
  64. 64.
    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 143:529–531CrossRefGoogle Scholar
  65. 65.
    Olson ME, Gard S, Brown M, Hampton R, Morck DW (1992) Flavobacterium indologenes infection in leopard frogs. JAVMA J Am Vet Med A 201:1766–1770Google Scholar
  66. 66.
    Taylor SK, Williams ES, Thorne ET, Mills KW, Withers DI, Pier AC (1999) Causes of mortality of the Wyoming toad. J Wildl Dis 35:49–57CrossRefPubMedGoogle Scholar
  67. 67.
    Densmore CL, Green DE (2007) Diseases of amphibians. ILAR J 48:235–254CrossRefPubMedGoogle Scholar
  68. 68.
    Geng Y, Wang KY, Zhou ZY, Li CW, Wang J, He M, Yin ZQ, Lai WM (2011) First report of a ranavirus associated with morbidity and mortality in farmed Chinese giant salamanders (Andrias davidianus). J Comp Pathol 145:95–102CrossRefPubMedGoogle Scholar
  69. 69.
    Becker MH, Harris RN (2010) Cutaneous bacteria of the redback salamander prevent morbidity associated with a lethal disease. PLoS One 5, e10957CrossRefPubMedPubMedCentralGoogle Scholar
  70. 70.
    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 16:807–820CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Obed Hernández-Gómez
    • 1
    Email author
  • Steven J. A. Kimble
    • 1
  • Jeffrey T. Briggler
    • 2
  • Rod N. Williams
    • 1
  1. 1.Department of Forestry and Natural ResourcesPurdue UniversityWest LafayetteUSA
  2. 2.Missouri Department of ConservationJefferson CityUSA

Personalised recommendations