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

2. Introduction Amphibians
Amphibians
Amphibian is derived from the Ancient Greek term amphíbios, which means both kinds of life, amphi meaning “both” and bio meaning life. Eventually it was used to refer to animals that live both in the water and on land.[5]
Amphibians-generally live in moist or aquatic environments.
They have mucous glands which produce an acidic secretion that helps prevent desiccation and promotes efficient O2 uptake. This secretion in rich in polysaccharides (very long chains of carbohydrates or sugars).
Because of the sugars the secretion can also act as a nutrient source for microorganisms.
So, amphibians are exposed to and attract many fungal and bacterial species, including pathogens that can cause disease.

3. Introduction
Massive decline in the number of amphibian species worldwide.
One disease is chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd).
Mycosis=fungal infection chytrid=order Chytridiales.
There have been massive declines in the numbers of amphibian species worldwide, and this is due to many factors:
There are six major factors negatively affecting amphibians, and all are due to human activity: habitat destruction, infectious diseases, pollution & pesticides, climate change, invasive species, and over-harvesting for the pet and food trades.
The disease has been proposed as a contributing factor to a global decline in amphibian populations that apparently has affected 30% of the amphibian species of the world.[1]
The chytrids have also been included among the Protista, but are now regularly classed as fungi.
Many chytrids are aquatic (mostly found in fresh water). There are approximately 1,000 chytrid species, in 127 genera, distributed among 5 orders.
Chytridiomycosis is believed to adhere to the following course: zoospores first encounter amphibian skin and quickly give rise to sporangia, which produce new zoospores.[4] The disease then progresses as these new zoospores reinfect the host. Morphological changes of amphibians infected with the fungus include a reddening of the ventral skin, convulsions with extension of hind limbs, accumulations of sloughed skin over the body, sloughing of the superficial epidermis of the feet and other areas, slight roughening of the surface with minute skin tags, and occasional small ulcers or hemorrhage. Behavioral changes can include lethargy, a failure to seek shelter, a failure to flee, a loss of righting reflex, and abnormal posture (e.g. sitting with the hind legs away from the body) [5]

4. Introduction
In the Southern San Joaquin valley, Bullfrogs (Rana catesbeiana) and California Toads (Bufo boreas halophilus) seem to be doing just fine!
R. catesbeiana
B. b. halophilus
Interestingly, it is not yet well understood why some amphibian species, even when infected do not succumb to the disease whereas others have died out in a very short time.
More specifically, in S S J valley…..
Non-native bullfrogs and toads seem to be thriving.
Kerry Kriger 2011
Charles M. Lane 2012

5. Introduction Hypothesis:
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.
Pretty ready known or excepted that skin bacteria of animals (including humans) acts as a protective barrier against invading microbial pathogens.
One reason for their success may be due to antifungal compound produced by cutaneous bacterial species.
The main purpose of the work I’m going to present is to determine if frogs/toads carry cutanenous antifungal producing bacteria that may be providing them with a competitive edge.

6. Rinse with sterile water
Methods
Catch frogs and toads
Rinse with sterile water
Swab frogs/toads
Streak plate
Project initiated in the summer of 2009-during chevron sponsored REVSUP
We began that 4-week program by going on a frog hunt. Specifically we were interested in catching bullfrogs and toads but we took this opportunity to catch any type of frog/toad. We collected frogs/toads from a variety of urban and rural locations. (Springs apartments, golf course, drainage canal, creek that feeds into Kern river).
How we caught them.
About 35 bullfrogs/26 toads
Frogs were rinsed with sterile water to remove any transient bacteria.
Swabbed 2X with sterile cotton swab released to their environment.
One swab streaked onto low nutrient R2A media plates, second was frozen for future use.
Plates incubated at room temp for ~2 weeks.
Growing collonies transferred to new plates to generate pure cultures. ~1700 isolates
Transfer to trypitcase soy yearst extract/glycerol -80 colony morphology documented
Scope to large 6/6 from one man-made pond location.
Purify cutaneous bacterial isolates
Fig. 1A
Fig. 1B
Fig. 1C
Fig. 1D
Fig. 1E
Fig. 1A
Fig. 1B
Fig. 1C
Fig. 1D
Fig. 1E

7. Purify fungal isolates Collect water from frog/toad environment
Methods/Results
Plate water samples
Purify fungal isolates
Collect water from frog/toad environment
DNA extraction from fungi
In order cultivate a variety of environmental fungal species. We also collected water samples, plated Sabouraud dextrose media and froze.
Agar plugs of Pure fungal mycelia were repeatedly transferred to new plates.
5 environmental fungi were isolated in this way.
We isolated DNA from the fungi using a comercially available kit. We then utilized a molecular method, PCR to amplify the 18S rRNA gene.
We set up “antifungal challenge assay”.
2 bacterial isolates are streaked in a thin line on a solid media plate with a piece(agar plug) of fungal mycelium in the center of each plate.
Incubate the plate for 4-14 days at room temp depending on the growth of the individual fungus.
Figure 1. Representative Challenge Assays. Challenge assays with unknown bacterial isolates against a fungus obtained from the amphibian environment on SAB medium. A clear inhibitory zone is visible for isolates #369 and #811, whereas no inhibition was observed for isolate #388 and #809.
Five fungi identified:
2 distinct Aspergillus sp.
Cochlibolus sp
Eupenicillium sp.
Galactomyces geotrichum
PCR amplification
DNA sequencing to identify fungi
Fig. 1A
Fig. 1B
Fig. 1C
Fig. 1D
Fig. 1E
Fig. 1A
Fig. 1B
Fig. 1C
Fig. 1D
Fig. 1E

8. Cutaneous bacteria challenged against environmental fungi
Methods
Cutaneous bacteria challenged against environmental fungi
In order cultivate a variety of environmental fungal species. We also collected water samples, plated Sabouraud dextrose media and froze.
Agar plugs of Pure fungal mycelia were repeatedly transferred to new plates.
5 environmental fungi were isolated in this way.
We isolated DNA from the fungi using a comercially available kit. We then utilized a molecular method, PCR to amplify the 18S rRNA gene.
We set up “antifungal challenge assay”.
2 bacterial isolates are streaked in a thin line on a solid media plate with a piece(agar plug) of fungal mycelium in the center of each plate.
Incubate the plate for 4-14 days at room temp depending on the growth of the individual fungus.
Figure 1. Representative Challenge Assays. Challenge assays with unknown bacterial isolates against a fungus obtained from the amphibian environment on SAB medium. A clear inhibitory zone is visible for isolates #369 and #811, whereas no inhibition was observed for isolate #388 and #809.
Fig. 1A
Fig. 1B
Fig. 1C
Fig. 1D
Fig. 1E
Fig. 1A
Fig. 1B
Fig. 1C
Fig. 1D
Fig. 1E

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
5 fungal species
Three classes of phylum Ascomycetes
Aspergillis- hundreds of species, common mold in damp basements. 60 different human disease
Cochlibolus- 55 speices many known as plant pathogens
Eupenicillium –soil fungi
Galactomyces geotrichum found lots of places early stage fruit some plumonary disease
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.

10. Challenge positive isolates against known pathogens
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.
Challenge positive isolates against known pathogens
Approach:
Infectious diseases remain one of the leading cuases of death in humans worldwide.
One approach to help combat these diseases to examine antibiotics from naturally occurring compounds.
Marine invertebrates
algae
Plants.
Turns out that some of these recent discoveries have strucutural similarities to know metabolites of microbial origin.

11. 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
It has a variety of names and is also known as thrush, athletes feet, vaginal yeast infection
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.
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

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.
Examine cutaneous bacterial diversity of frogs and toads.
bioweb.uwlax.edu 2008
Impactlab.com 2009
flickriver.com 2012

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