Antifungal Properties of Cutaneous Bacteria Found on Rana catesbeiana (North American Bullfrog) and Bufo boreas halophilus (California Toad).

Kathy Szick-MirandaCalifornia State University, Bakersfield

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

http://www.californiaherps.com/salamanders/images/avagranshu04.jpg

Amphibians

Introduction

http://4.bp.blogspot.com/_9py6IgNlFak/TBbDsk1BIQI/AAAAAAAAAdY/R9Ny0PfOPQU/s1600/bullfrog.jpg

http://frogspictures.net/California-Toad.jpg

http://3.bp.blogspot.com/-3fHERaN40H4/Tc1frxaD5xI/AAAAAAAAAhI/HkEBZF5HGBg/s1600/newts+3+of+them.jpg

http://do-while.com/img/life/colorful-frogs/colorful-frogs02.jpg

Massive decline in the number of amphibian species worldwide.

One disease is chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd).

Introduction

http://amphibiaweb.org/images/dead_rana_muscosa_small.jpghttp://theworldofrogs.weebly.com/uploads/3/0/6/2/3062899/8103653_orig.jpg?335

In the Southern San Joaquin valley, Bullfrogs (Rana catesbeiana) and California Toads (Bufo boreas halophilus) seem to be doing just fine!

Introduction

R. catesbeiana B. b. halophilus

www.calris.org Charles M. Lane 2012http://savethefrogs.com Kerry Kriger 2011

http://www.power-technology.com/projects/san_joaquin/images/sjvec.jpg

Cutaneous bacteria act as a protective barrier.

Marsh and Selwyn 1977; Al-Admawy and Noble 1981; McFall-Ngai et al. 2005

Cutaneous bacteria of some amphibians produce antibiotics that protect their hosts from pathogenic fungi.

Austin 2000; Brucker et al. 2008a, 2008b; Harris et al. 2009

Introduction

Hypothesis:Bullfrogs and California Toads possess cutaneous bacteria that will inhibit the growth of some fungi.

Catch frogs and toads

Streak plate

Purify cutaneous bacterial isolates

Methods

Rinse with sterile water

Swab frogs/toads

DNA extraction from fungi

PCR amplification

DNA sequencing to identify fungi

Methods/Results

Five fungi identified:

2 distinct Aspergillus sp.

Cochlibolus spEupenicillium sp.Galactomyces

geotrichum

Plate water samples

Purify fungal isolates

Collect water from frog/toad environment

Methods

Cutaneous bacteria challenged against environmental fungi

Results233 pure bacterial isolates challenged against 5 environmental fungi

5 isolates were positive against 4 fungi5 isolates were positive against 3 fungi16 isolates were positive against 2 fungi43 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.Galatomyces geotrichum inhibited by 22% of the positive

isolatesAspergillus sp. (b) inhibited by 9% of the positive isolates.

Question

Hypothesis #2: Bullfrogs and California Toads possess cutaneous bacteria that will inhibit the growth of known amphibian and human pathogens.

Data support the original hypothesis.

Challenge positive isolates against known pathogens

Approach:

Pathogens• Basidiobolus ranarum: • 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

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

ConclusionsBullfrogs and toads possess cutaneous bacteria that inhibit the growth of some fungi.

Some cutaneous 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

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.

Examine cutaneous bacterial diversity of frogs and toads.

flickriver.com 2012bioweb.uwlax.edu 2008 Impactlab.com 2009

Future Work

• 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

Acknowledgements