Antifungal Properties of
Cutaneous Bacteria Found
on Rana catesbeiana (North
American Bullfrog) and Bufo
boreas halophilus
(California Toad).
Kathy Szick-Miranda
California State University, Bakersfield
www.grit.com/animals/wildlife/american-bullfrog.aspx 2010
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/colorfulfrogs/colorful-frogs02.jpg
http://3.bp.blogspot.com/3fHERaN40H4/Tc1frxaD5xI/AAAAAAAAAhI/HkEBZF5HGBg/s160
0/newts+3+of+them.jpg
http://www.californiaherps.com/salamanders/images/avagr
anshu04.jpg
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.j
pg?335
Introduction
In the Southern San Joaquin valley,
Bullfrogs (Rana catesbeiana) and
California Toads (Bufo boreas
halophilus) seem to be doing just
fine!
http://www.powertechnology.com/projects/san_joaquin/ima
ges/sjvec.jpg
R. catesbeiana
B. b. halophilus
http://savethefrogs.com Kerry Kriger 2011
www.calris.org Charles M. Lane 2012
Introduction
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
Hypothesis:
Bullfrogs and California Toads possess cutaneous bacteria
that will inhibit the growth of some fungi.
Methods
Catch frogs and toads
Rinse with sterile water
Swab frogs/toads
Streak plate
Purify cutaneous bacterial isolates
Methods/Results
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.
Galactomyces geotrichum
PCR amplification
DNA sequencing to identify fungi
Methods
Cutaneous bacteria challenged
against environmental fungi
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.
Galatomyces geotrichum inhibited by 22% of the positive isolates
Aspergillus sp. (b) inhibited by 9% of the positive isolates.
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
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
Conclusions
Bullfrogs 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
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.
Examine cutaneous bacterial diversity of frogs and toads.
bioweb.uwlax.edu 2008
Impactlab.com 2009
flickriver.com 2012
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