Transcript Slide 1

Sponge squeeze
How sanitary can a stinky sponge be?
An analysis of antimicrobial activity in Gulf of Maine
specimen of the Halichondria sp. marine sponge
Françoise Morison
Department of Chemistry, Inco 590, University of New Hampshire, Manchester
Abstract
Conclusion
Many of the chemical substances found in marine sponges are
bioactive and research on secondary metabolites is being pursued
in the fields of drug discovery and chemical ecology. Specimen of
Halichondria sp., a marine sponge commonly found in the waters
of the Gulf of Maine, were investigated for their production and
bioactivity of such compounds. Three of the purified fractions
produced in this study showed to inhibit growth of the gram (-)
bacterium Pseudomonas aeruginosa.
Introduction
Marine sponges are chemical factories that are sources of potential
pharmaceuticals(1). The isolation by Werner Bergman of two
marine sponge nucleosides in the early 1950’s led to the synthesis
of the anti-HIV drug AZT and the anti-herpes drug Acyclovir (2).
Since then, thousands of new compounds have been reported
(1,3). Many have bioactivity against cancer, pathogenic viruses,
bacteria, and fungi (1). Some are currently undergoing clinical
trials (4).
The ecological function of these metabolites is poorly understood.
Their role in feeding deterrence, allelopathy, and control of
bacterial attachment have been described (5,6). It is speculated
that some sponge metabolites may in fact be synthesized by
symbiotic microorganisms that can represent up to 40% of the
sponge biomass (7). Little is known of the nature of these
associations.
Two species of marine sponges of the genus Halichondria are
commonly found in the Gulf of Maine. Both are described as
having a sulfur odor, which suggests potential biosynthesis of
toxic chemicals.
A search of the Napralert database determined that among
marine sponges occurring locally, species of Halichondria were the
most studied, but all studies were done abroad. (8). A variety of
steroids were isolated from H. bowerbanki by an Italian group (8).
Extracts from both H. panicea and H. bowerbanki that were tested
by Andersson et al. (Sweden) for antimicrobial activity were
reported inactive (8).
Materials and Methods (cont’d)
Extraction and purification
Bioassay
The fresh sponge samples (760 g, wet wt) were extracted with
methanol (1 L x 3). After concentration, the methanol crude
extract (300 ml) was submitted to solvent-solvent partitioning
into hexane, ethyl acetate, and methylene chloride phases
successively. The ethyl acetate and methylene chloride extracts
were chromatographed on a silica gel column using a mixture of
30% hexane 70% ethyl acetate as eluent.
Four ethyl acetate fractions were tested.
Trypticase soy agar plates were swabbed with
Pseudomonas aeruginosa , Proteus mirabilis, and
Staphylococcus aureus. The extracts were tested
using the sterile filter paper disk method. A blank
disk and two standard antibiotic disks
(tetracycline 30μg and chloramphenicol 30μg)
were used as control.
Some of the results supported the hypothesis that Halichondria sp.
may produce toxic substances Further study is needed to try and
resolve the uncertainties mentioned in the results, i.e.
concentration of the extracts and solvent involvement in toxicity.
Sufficient amount of compound needs to be extracted to evaluate
its concentration. Solvent control volume and time of evaporation
must systematically be measured.
NMR analysis may be pursued in an attempt to determine the
structure of the active compound. Structure elucidation may
however require further purification.
Extraction and culture of bacteria or separation of microalgae
possibly present in the sponge may provide additional
information as to the source of the active substance(s).
Literature cited
Results
Three of the fractions obtained from the ethyl acetate extract
showed some inhibition of Pseudomonas aeruginosa. Inhibition was
observed on that bacterial species only. Some zones had an
irregular shape.
These fractions were eluted from the column successively and
may therefore contain the same compound.
While results of this study do not support what had been
reported by Andersson et al., the group had tested different
extracts (H2O, Pet. Ether, CHCl3, and methanol) (8).
Because the extract concentrations could not be calculated, due to
minimal immeasurable amount of product extracted, it is not
possible to assess the significance of the zone of inhibition
observed. Furthermore, whether inconclusive zones of inhibition
observed on some plates are related or not to possible toxicity of
lingering solvent remains unclear. No inhibition was observed
when solvent testing was repeated.
Table 1.
Three fractions inhibited the growth of P. aeruginosa.
P. aeruginosa 1
P. mirabilis
S. aureus
_______________________________________________________________
FM2-I-(2-3)
FM2-II-(4)
FM2-III-(5)
FM2-IV-(6)
Tetracycline*
Chloramphenicol*
Inhibited
Inhibited
Inhibited
Negative
Resistant
Resistant4
Inconclusive2
Negative
Inconclusive2
Negative
Resistant3
Sensitive
1-Notorious for its resistance to antibiotics
2-Inhibition ring present around solvent control
3-Reported occurrence of R-plasmid in strains of P. mirabilis
Negative
Negative
Negative
Negative
Sensitive
Sensitive
4- expected
*Reported antimicrobial sensitivity are based on the guidelines of the Kirby-Bauer method
(1995).
Fraction FM2-I-(2-3) with
ring of inhibition
Fraction FM2-II-(4) showing inhibition.
Note irregularity around one disk.
Materials and Methods
1. Bhakuni D.S., Rawat D.S. Bioactive Marine Natural Products. New York:
Springer; 2005. 382 p.
2. Harbor Branch Oceanographic Institution Media Lab website. The pipeline
and the finish line: the first wave of marine-derived drugs.
<http://www.marinebiotech.org/pipeline.html#w1>
3. Kornprobst Jean-Michel.. Les medicaments de la mer. Faculté de Pharmacie
et Institut Substances et Organismes de la mer (ISOmer) Université de Nantes.
2001.
4. Simmons T. L., Andrianasolo E. , McPhail K., Flatt P. , Gerwick W. H. Marine
natural products as anticancer drugs. Molecular cancer therapeutics [online]
2005; 4(2): 333-342. Avail. From: http://mct.aacrjournals.org/cgi/reprint/4/2/333.
5. Kelly S.R., Jensen P.R., Henkel T.P., Fenical W., Pawlik J.R. Effects of
Caribbean sponge extracts on bacterial attachment. Aquatic microbial ecology
2003; 31:175-182.
6. Assmann M., Lichte E., Pawlik J.R., Kock M. Chemical defenses of the
Caribbean sponge Agelas wiedenmayeri and Agelas conifera. Marine ecology
progress series 2000; 207:255-262.
7. Guyot Michele. Intricate aspects of sponge chemistry. Zoosystema 2000;
22(2).
8. Napralert. Program of collaborative research in the pharmaceutical sciences.
College of Pharmacy. University of Illinois at
Chicago.<http://www.napralert.org/>
Collection of animal material
A total of 6 organisms of the sponge genus Halichondria were
collected on March 20, 2007, within a 50 yard radius at a depth of
3 feet off Laighton Point, Pembroke, Maine (44o55’N;66o55’W).
Water temperature was 36oF; the water salinity was 32 ppt.
Area of collection
Blank disk with solvent
Halichondria specimen
Chloramphenicol (30μg)
(no ring of inhibition)
Acknowledgements
I want to thank Dr. Lorraine Doucet, Dr. Sarah Kenick, Dr. Stephen Pugh,
Professor Allan Ray, and Keith Legro, for their help, advice and support. I
also want to acknowledge Dr. William Sponholtz from Cushing Academy, Ma.,
for sharing methods used in his own research. Thank you to Dr. Richard
Johnson from the Durham Chemistry department for lending the equipment.
Thanks Jeremy Neal for sharing the dishwashing.
For further information
Tetracycline (30μg) with
small ring of inhibition
(<14 mm)
Please don’t hesitate to contact the author at [email protected]