www.grit.com/animals/wildlife/american-bullfrog.aspx 2010

1. Antifungal Properties of Cutaneous Bacteria Found on Ranacatesbeiana(North American Bullfrog) and Bufoboreashalophilus (California Toad). Kathy Szick-Miranda California State University, Bakersfield www.grit.com/animals/wildlife/american-bullfrog.aspx 2010

2. Introduction Amphibians http://4.bp.blogspot.com/_9py6IgNlFak/TBbDsk1BIQI/AAAAAAAAAdY/R9Ny0PfOPQU/s1600/bullfrog.jpg http://frogspictures.net/California-Toad.jpg http://do-while.com/img/life/colorful-frogs/colorful-frogs02.jpg http://3.bp.blogspot.com/-3fHERaN40H4/Tc1frxaD5xI/AAAAAAAAAhI/HkEBZF5HGBg/s1600/newts+3+of+them.jpg http://www.californiaherps.com/salamanders/images/avagranshu04.jpg

3. Introduction Massive decline in the number of amphibian species worldwide. One disease is chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). http://amphibiaweb.org/images/dead_rana_muscosa_small.jpg http://theworldofrogs.weebly.com/uploads/3/0/6/2/3062899/8103653_orig.jpg?335

4. Introduction In the Southern San Joaquin valley, Bullfrogs (Ranacatesbeiana) and California Toads (Bufoboreashalophilus) seem to be doing just fine! http://www.power-technology.com/projects/san_joaquin/images/sjvec.jpg R. catesbeiana B. b. halophilus http://savethefrogs.com Kerry Kriger 2011 www.calris.org Charles M. Lane 2012

5. Introduction Cutaneous bacteria act as a protective barrier. Marsh and Selwyn 1977; Al-Admawy and Noble 1981; McFall-Ngaiet al. 2005 Cutaneous bacteria of some amphibians produce antibiotics that protect their hosts from pathogenic fungi. Austin 2000; Bruckeret al. 2008a, 2008b; Harris et al. 2009 Hypothesis: Bullfrogs and California Toads possess cutaneous bacteria that will inhibit the growth of some fungi.

6. Methods Fig. 1A Fig. 1A Fig. 1B Fig. 1B Fig. 1C Fig. 1C Fig. 1D Fig. 1D Fig. 1E Fig. 1E Catch frogs and toads Rinse with sterile water Swab frogs/toads Streak plate Purify cutaneous bacterial isolates

7. Methods/Results Fig. 1A Fig. 1A Fig. 1B Fig. 1B Fig. 1C Fig. 1C Fig. 1D Fig. 1D Fig. 1E Fig. 1E Collect water from frog/toad environment Plate water samples Purify fungal isolates DNA extraction from fungi Five fungi identified: 2 distinct Aspergillus sp. Cochlibolus sp Eupenicillium sp. Galactomycesgeotrichum PCR amplification DNA sequencing to identify fungi

8. Methods Fig. 1A Fig. 1A Fig. 1B Fig. 1B Fig. 1C Fig. 1C Fig. 1D Fig. 1D Fig. 1E Fig. 1E Cutaneous bacteria challenged against environmental fungi

9. Results 233 pure bacterial isolates challenged against 5 environmental fungi 5 isolates were positive against 4 fungi 5 isolates were positive against 3 fungi 16 isolates were positive against 2 fungi 43 isolates were positive against 1 fungus Additional results: Cochlibolus sp. inhibited by 87% of the positive isolates. Eupenicillium sp. inhibited by 20% of the positive isolates. Aspergillus sp. (a)inhibited by 20% of the positive isolates. Galatomycesgeotrichuminhibited by 22% of the positive isolates Aspergillus sp. (b)inhibited by 9% of the positive isolates.

10. Question Data support the original hypothesis. Hypothesis #2: Bullfrogs and California Toads possess cutaneous bacteria that will inhibit the growth of known amphibian and human pathogens. Approach: Challenge positive isolates against known pathogens

11. Pathogens • Basidiobolusranarum: • known human and amphibian pathogen • causes skin and GI lesions • found worldwide • Candida albicans: • normally found in low levels in the human body • causes yeast infections • Cryptococcus neoformans: • affects immunocompromised patients • causes lung infections

12. Results • 85% of isolates inhibited the growth of B. ranarum • 39% of isolates inhibited the growth of C. albicans • 76% of isolates inhibited the growth of C. neoformans

13. Conclusions Bullfrogs and toads possess cutaneous bacteria that inhibit the growth of some fungi. Somecutaneous bacteria isolated from bullfrogs and toads inhibit the growth of known pathogenic fungi. •  Implications: • Improvement in amphibian conservation • Advances in the treatment of fungal pathogens

14. Future Work • Complete challenge assays with known pathogens. • Identify positive bacterial isolates. • Challenge the positive isolates against Bd and other known human pathogens. • Determine which metabolites in each of the bacterial species exhibit • antifungal activity. • Examinecutaneous bacterial diversity of frogs and toads. flickriver.com 2012 bioweb.uwlax.edu 2008 Impactlab.com 2009

15. Acknowledgements • Dr. Antje Lauer – CSUB • Chevron REVS-UP Program – CSUB • CSUPERB-Faculty Seed Development Grant • Chevron REVS-UP participants 2009-2011 • Student Researchers: • Amanda Payne • Ashley Nunez • Lauren Dowel • Christine Hluza • David Tate • Kathryn Hubert • Esther Ibarra • RJ Jimenez scientificamerican.com 2011