2004

Asiatic Herpetological Research

Vol.10, pp. 161-163

Antimicrobial Activity in the Skin Secretion of Bufo viridis (Laurenti, 1768)
BA şARAN DÜLGER1,*, İSMAIL HAKKI UĞURTAş 2, AND MURAT SEVINÇ2
1

Canakkale Onsekiz Mart University, Faculty of Science & Arts, Department of Biology, Canakkale-Turkey
2Uluda ğ University, Faculty of Science & Arts, Department of Biology, Bursa-Turkey*Correspondence author, E-mail: dbasaran@comu.edu.tr; Fax number: +90.286.2180533

Abstract. - In this study, antimicrobial activity of various extracts prepared from Bufo viridis skin secretion weretested against the microorganisms by disk diffusion method. Escherichia coli ATTC 10536, Listeria monocytogenes
ATCC 19117, Klebsiella pneumoniae UC57, Salmonella typhi ATCC 19430, Staphylococcus aureus ATCC 6538P,
Mycobacterium smegmatis CCM 2067, Rhodotorula rubra and Saccharomyces cerevisae ATCC 9763 were used.
According to our results, the extracts prepared from Bufo viridis skin secretion have high antimicrobial activity
against the tested microorganisms.
Key words. - Bufo viridis, Amphibia, antimicrobial activity, skin secretion.

Introduction
Amphibians have skin glands producing mucous andpoison. Amphibians have been studied and haveattracted special attention from a toxicological point ofview. Various substances with antimicrobial activity
have been isolated from skin secretions of amphibianspecies (Dapson, 1970; Croce et al., 1973; Dapson et al.,
1973; Preusser et al., 1975; Cevikbas, 1978). Several
toxins in amphibian poisons have been used as experimental tools and contributed to significant progress inphysiology. Some toxins (Batrachotoxins) specifically
block the inactivation of the voltage regulated Na+ channels in nerve and muscle cells, which causes a massive
inflow of Na+. The cells become irreversibly depolarized, which, among other things, produces heart arrhythmia and respiratory failure and finally cardiac
insufficiency. In humans, some amphibian toxins (Bufotenin) produce symptoms similar to those of LSD (Lutz,1971; Edstrom, 1992). In previous studies, some skinsecretions showed remarkable cytotoxic activity against
eukaryotic cells (Kolbe et al., 1993; Sanna et al. 1993).
The aim of the this study is to test the antimicrobialactivity of Bufo viridis skin secretions against Grampositive, Gram-negative bacteria and yeast cultures for
future possible use in providing pharmacological tools
for the study of new drugs and aid in benefitting humanhealth.

Materials and Methods
Specimens of Bufo viridis were collected from different
regions in Bursa, Turkey in March 1998. Collected frogs© 2004 by Asiatic Herpetological Researchwere brought to the laboratory and kept in an aquarium.
Before experimentation, the frogs were washed firstwith tap water and then with distilled water. They wereplaced for 3-5 minutes in a glass jar containing a pieceof cotton soaked with ether to stimulate skin secretions.
The secretion accumulated on the skin was obtained byscraping the body of the animals with a spatula. The
foamy secretion thus obtained was placed in a tube, leftin an 80°C water bath for 30 min and centrifuged at
5,500 rev/min for 30 min. After centrifugation, the precipitate was used in the experiments. Before using in theexperiments, the precipitate was diluted with distilledwater 0.1 M HCl, 0.1 M NH4OH, and 1 M phosphatebuffers (pH: 4 and pH: 7).
In this study, Escherichia coli ATTC 10536, Listeria monocytogenes ATCC 19117, Klebsiella pneumoniae UC57, Salmonella typhi ATCC 19430,
Staphylococcus aureus ATCC 6538P, Mycobacterium
smegmatis CCM 2067 bacteria cultures and Rhodotorula rubra and Saccharomyces cerevisae ATCC 9763
yeast cultures were used.
In vitro antimicrobial activity studies were carriedout by the Agar-Disc Diffusion Method. Mueller Hinton
Agar (Oxoid) was preferred as the most suitablemedium for antimicrobial activity studies. Each extract
was implemented into a sterile disc in varying concentrations starting from 20 µl. Each disc was 6 mm indiameter.
Bacteria and yeast cultures were suspended in 4-5
ml Brain Heart Infusion Broth (Oxoid) and Malt Extract
Broth (Difco). Bacteria were incubated in 37°C for 2-5hours. Yeast cultures were incubated in 30°C for 5-7hours. A visible turbidity was obtained at the end of thistime. The turbidity of bacterial suspension was adjusted

Vol.10, p. 162

Asiatic Herpetological Research

2004

Table 1. Antimicrobial activity of various extracts of Bufo viridis skin secretions on microorganisms.
Phosphate
0.1 N

0.1 N

Distilled

Microorganisms / solvents

BufferHClNH4OH

water
pH: 4

pH: 7

Escherichia coli ATCC 10356

++

+++

(+)

(+)

++

Listeria monocytogenes ATCC 19117

+++

+++

++

++

++

Staphylococcus aureus ATCC 6538P

+++

++

++

++

+++

Klebsiella pneumoniae UC57

+++

++

+++

++

+++

Salmonella thyphi ATCC 19430

+++

++

+++

++

++

Mycobacterium smegmatis CCM 2067

+++

+++

++

++

+++

Rhodotorula rubra

+++

+++

+++

+++

++

Saccharomyces cerevisiae ATCC 9730+++++++++++++++(+) : Inhibition zone less than 1 mm surrounding the 6 mm paper disk.
+ : Inhibition less than++ : Inhibition comparable to+++: Inhibition more than 10 µg penicillin or sulconazole / disk; Inhibition zones of references : 12-16 mm diameter.

according to Macfarland Standard Tube [0,5] with physiologic serum and inoculation performed. Prepared bacterial suspension was mixed with a sterile applicator andexcess fluid of applicator was removed by rotating theapplicator to one side of the tube. We streaked the entire
Mueller Hinton Agar surface in three different directions by rotating the plate 60° angles after each streaking. Yeast cultures were inoculated into Muller Hinton
Agar (102 cfu/ml). All petri dishes after inoculation wereallowed to dry for 15-20 min at room temperature (bacteria at 35°C and yeast at 30°C). Inhibition zone diameters were measured after 24-48 hours (Collins et al.
1987, NCCLS 1993). In addition, continued only solvent was used as negative control disc and antibiotic
penicillin and sulcanozole discs were used as references.
Experiments were repeated three times and results wereexpressed as average values.

Results and Discussion
Antimicrobial activity effects of five different extracts,
which were prepared by using distilled water, 0.1 NHCl, 0.1 N NH4OH, 1 M phosphate buffers (pH: 4 andpH: 7), were obtained from the skin secretions of Bufo
viridis against bacteria and yeast cultures, results aregiven in Table 1.
According to our findings, all the extracts of skinsecretion against the yeast cultures exhibit higher antimicrobial activity than that of a compared antibiotic.

The 0.1 M HCl extract shows more effect than that ofthe other extracts against bacteria. 1 M phosphate buffer(pH: 4 and 7) extracts exhibited minor effects against
Escherichia coli. However, phosphate buffer (pH: 4 and7) extracts exhibited strong effects against the other bacteria. It can be said that the active substance obtained
from Bufo viridis skin secretion dissolves easily in the0.1 M HCl and has high antimicrobial activity as a consequence. It has been reported that sensitivity of themicroorganisms to the chemotherapeutic agentschanges from strain to strain (Cetin et al., 1989). Our
results are in agreement with the other authors’ results.
Inhibition zone diameters around the control discwere measured as 0-1 mm. In this study, antimicrobial
effects of the prepared extracts on the tested microorganisms were determined by using different solvents.
Croce et al. (1973) investigated antimicrobial activity of skin secretions from Bombina variegata pachypus. They homogenized skin secretion with phosphatebuffers (pH: 4 and 7) 1 M HCl, 1 M NH4OH and distilled water. These homogenates show high antimicrobial activity against Staphylococcus aureus but they do
not show any antimicrobial effect against Aspergillus
niger, Trichophyton mentagrophytes ATCC 8757 andCandida albicans.
Cevikbas (1978) examined antibacterial activity inthe skin secretions of Rana ridibunda. The author
reported that skin secretion of Rana ridibunda shows
antibacterial activity at different levels. However, in our2004

Asiatic Herpetological Researchpresent study, skin secretions of Bufo viridis against theyeast cultures shows more antimicrobial activity thanthat of the bacterial cultures. Our findings parallel thosereported in the above studies. In Amphibia, antimicrobial activity of skin secretions differ at both the genericand specific levels.
Although the antimicrobial activity of skin secretions from Bufo viridis, Bufo vulgaris, Salamandra maculosa and Salamandra atra were determined (Pavan,1962; Pavan and Nascimbene, 1948), antimicrobialactivity of skin secretion from Bufo marinus, Triturus,and Xenopus were not observed (Preusser et al., 1975;
Kolbe et al., 1993; Ozeti and Yilmaz, 1994). Antiyeast
activity observed in our study was not observed in
Croce’s et al. study (1973). Our results show that skinsecretion components from Bufo viridis may be different
from Bombina variegata pachymus.

Acknowledgments
The authors are grateful to Aegean University, Science
Faculty, Department of Biology, and Basic and Industrial Microbiology for supplying the strain of microorganism used in the study.

References
Cetin, T. E. and N. Gurler. 1989. Bakterilerin Antibiyotiklere Duyarlilik Deneyinin Yapilmasi. Kukem
12:2-5.
Cevikbas, A. 1978. Antibacterial activity in the skinsecretion of the frog Rana ridibunda. Toxicon
16(2):195-197.
Collins, C. M., P. M. Lyne, and J. M. Grange. 1989.
Microbiological Methods, Six Edition. Butterworths & Co. Ltd., London. 410 pp.
Croce, R., N. Giglioli, and L. Bolognani. 1973. Antimicrobial activity in the skin secretions of Bombina
variegata pachymus. Toxicon 11:99-100.
Edstrom, A. 1992. Venomous and Poisonous Animals.
Kriger Publishing Company. Malabar, Florida.
USA. 226 pp.
Kolbe, H. V. I., A. Huber, P. Cordier, U. B. Rasmussen,B. Bouchon, M. Jaqunod, R. Vlasak, E. Delot, andG. Kreil. 1993. Xenoxins: A Family Of Peptides
From Dorsal Gland Secretion Of Xenopus laevis
Related To Snake-Venom Cytotoxins And Neurotoxins. Journal of Biological Chemistry268(22):16458-16464.

Vol.10, p. 163

Lutz, B. 1971. Venomous Animals and Their Venom.
Academic Press, London. 423-427 pp.
NCCLS. 1993. Performance Standards for Antimicrobial Disk susceptibility Tests. Approved Standard
NCCLS publication M2- A5, Villanova, PA, USA.

Özeti, N. and İ. Yilmaz. 1994. [Amphibians of Turkey].
Aegean University Press, İ zmir. 151 pp.
Pavan, M., 1962. Die Antibiotica tierischer Herkunft.
Zeitschrift Fur Hygiene und Infektionskrankheiten
34:136-138.
Pavan, M., and A. Nascimbene. 1948. Studi sugli antibiotici di origine animale. Bolletino Societa Medicochirurgica, Pavia 1-2:229.
Preusser, H. J., G. Habermehl, M. Sablofski, and H. D.
Schmall. 1975. Antimicrobial activity of alkoloid
from amphibian venoms and effects on the ultrastucture of yeast cells. Toxicon 12: 285.
Sanna, A. P., A. Barnabei, and G. Delfino. 1993. The
Cutaneous Venom of Bombina orientalis: CytotoxicEffects on the human HL 60 Cell Line and A Comparison with Bombina variegata. Journal of NaturalToxins 2(2):161-173.