Proc. West. Pharmacol. Soc 50: 152-155 (2007)
Screening of Antimicrobial Activity of Marine Sponge Extracts Collected from Tunisian Coast
Touati Ines1, Bakhrouf Amina2, Said Khaled1 and Gaddour Kamel1*
Laboratoire de Génétique, Biodiversité et Valorisation de Bioressources. Institut Supérieur de Biotechnologie de Monastir, Tunisie;
Laboratoire d’Analyses et de Contrôle des Polluants Chimiques et Microbiologique de l’Environnement,
Faculté de Pharmacie, Monastir, Tunisie
The antibacterial activity of the ethyl acetate extracts Among marine organisms, sponges are considered
of seven marine sponges collected from the Tunisian interesting targets to screen for antimicrobial
Mediterranean coast (Monastir) were tested against substances for many reasons. In fact these animals
eight human pathogenic bacteria and six human are frequently exposed to intense predation and
pathogenic fungi using the agar disk diffusion tissue infection by micro organisms . However,
method. The results show that 90% of the sponge despite being sessile and soft bodied, sponges
extracts present significant activity against at least appear to be predated only by selected groups of
one bacterial strain. Extracts of the sponges Agelas marine animals, such as turtles , sea urchins 
oroides and Axinella damicornis appeared to be quite and sea stars . While sponge tissue and skeletal
promising due to their capacity to inhibit the growth of constituents appear to have little or no activity against
Pseudomonas aeruginosa and gentamycin resistant potential predators , sponge secondary metabo-
strains of Listeria monocytogenese and Enterococcus lites clearly present a defensive role against predation
feacalis as well as broad spectrum activity against all . Sponges are the most primitive invertebrates, and
the other bacteria. The antifungal activity of these represent an important constitutive group of the coral
sponge extracts is not so promising, in fact only three reef fauna with a wide range of species .
among 9 sponge extracts show moderate capacity of
A variety of antimicrobial substances have been
growth inhibition against fungi strains. Agelas oroides
isolated from various species of marine sponges .
which shows interesting antibacterial activity, has
Up to 800 antibiotic compounds have been isolated
moderate activity against fungi strains tested in this
from marine sponges, a number that corroborates
study. Our results with antibacterial and antifungal
assumptions that sponges appear to defend
activity in vitro open the way for complementary
themselves against infections by producing and/or
investigation in order to purify and identify active
accumulating secondary metabolites.
In this report, we describe the biological effect of ethyl
acetate extracts of seven marine sponges collected
With a wide range of vegetable, animal and microbial
from the coastline of Tunisia for their antimicrobial
species, the sea is a nearly inexhaustible source of
activities against eight human pathogenic bacteria
new active natural substances. For many years,
and six human pathogenic fungi in order to find new
scientists have collaborated to study the biological
antibacterial and antifungal metabolites. For such, we
activity of marine organism extracts, helped by
tested the antimicrobial activity of the sponge extracts
sophisticated techniques of extraction, separation,
using several bacterial and fungal species, including
and analysis. The majority of antimicrobial activities
resistant strains such as Pseudomonas aeruginosa.
result from their cytotoxicity against micro organisms.
Indeed, many organisms have developed a chemical MATERIALS AND METHODS
Biological material - Sponge samples were collected by scuba
strategy for defence to ensure their survival, and to
during scientific expedition in a rocky slope at water deeper than
synthesize extremely active molecules that must act 20 m from the coastal water of Monastir, Tunisia (March 2006).
in a diluting aqueous environment. Therefore, some Specimens were cleaned and stored at -20°C until used in
marine invertebrates, especially sessile forms, extraction.
produce species-specific antifouling compounds  Preparation of extracts - Each sponge sample (400g wet weight)
and with increased use of antibiotics or chemical was cut into small pieces and macerated at 4°C during 48 hr.
The lyophilised material was subjected to an extraction of
substances, their use is becoming restricted not only biologically active components which were carried out with
due to the emergence of drug-resistant forms but also different solvents in the order of increase polarity: hexane, ethyl
for their adverse effects on the ecosystem . A acetate and methanol by soaking at ambient temperature. The
decreased efficacy of antibiotics, regardless of their residues (crude extracts) thus obtained were finally dried under
mechanism of action, leads to the need for suitable rotary vacuum evaporator (Heidolph) and screened against eight
human pathogen bacteria and six human pathogen fungi using
alternatives. the agar disk diffusion method.
Screening for antibacterial activity of sponge extracts - Bacterial against M. luteus. The inactivity towards these
strains: The reference strains of human pathogens used to test bacteria that we observed could be probably due to
antimicrobial activity included Gram-positive cocci: Staphylo-
coccus epidermidis Collection Institute Pasteur 106510, the developmental stage of the animals.
Staphylococcus aureus ATCC 25923, Micrococcus luteus NCIMB As shown in Table I, ethyl acetate extracts from
8166, Enterococcus feacalis ATCC 29212, and Gram-negative
bacteria: Escherichia coli ATCC 35218, Pseudomonas Axinella damicornis exhibited significant activity
aeruginosa ATCC27853, Salmonella thyphymerium LT2 and against all bacterial strains. An important activity
Listeria monocytogenese ATCC 19115. against the bacterial strains is also observed with the
The antibacterial activity of sponge extracts was performed using organic extract of Agelas oroides although it showed
the agar-disk diffusion assay . The bacterial cultures were first weak activity against Enterococcus feacalis, Listeria
grown on Muller Hinton infusion agar (MHI) plates at 37°C for 18 Monocytogenes and Pseudomonas arugenosa. All
to 24 hr prior to seeding onto nutrient agar. One or several the other organic extracts were active at least against
colonies of similar morphology of the respective bacteria were
transferred into API suspension medium (Biomérieux) and one of the bacterial strains.
adjusted to the 0.5 McFarland turbidity standard with a Densimat Table I Antibacterial activity of the marine sponge ethyl acetate extracts
(Biomérieux). The inocula of the respective bacteria were
streaked into the MHI agar plates using a sterile swab and were Marine Sponges Bacterial Species
then dried. A sterile filter disk 6 mm in diameter (Whatman paper
S. aureus S. epidermidis M luteus E coli
N°. 3) was placed on the infusion agar seeded with bacteria and ATCC2592 CIP106510 NCIMB8166 ATCC35218
ten 10 μL of the extract was dropped onto each paper disk A. Oroides 17 13 18 13
(5 mg/disk). The treated Petri dishes were kept at 4°C for 1 hr and A damicornis 17 21 26 20
were incubated at 37°C for 18 to 24 h. The antibacterial activity
P ficiformis 8 0 7 0
was evaluated by measuring the zone of growth inhibition
I. sarcotragus 7 0 0 0
surrounding the disks. Standard disks of the antibiotic
Gentamycin (10 μg) served as the positive antibacterial controls I spinosula 7 7 0 7
according to CASFM 2005 guidelines. H. communis 7 10 7 0
Spongia sp. 8 9 9 0
Screening for antifungal activity of sponge extract - Fungal
strains: The human pathogenic fungi used in this study were Gentamycin 10 UI 22 8 25 18
isolated from patient-associated infection. The strains used were E. feacalis L monocytogene P aerugenosa S thyphymerium
Candida albicans, Candida krusei, Candida tropicalis, Candida ATCC29212 ATCC19115 ATCC278 LT2
parapsilosis, Candida glabrata and Candida dubliniensis. These A. Oroides 8 9 8 12
strains were isolated on Sabouraud chlorenphenical plate and A damicornis 20 20 12 16
identified with the Api ID32C system (Biomerieux, France)
P ficiformis 0 0 7 7
according to the manufacture’s recommendation.
I. sarcotragus 0 0 0 0
Screening for the antifungal activity of the extracts was performed I spinosula 0 0 8 0
using the agar-disc diffusion method as previously described . H. communis 0 0 0 0
All fungal cultures were first grown on Sabouraud chlorenphenical
Spongia sp. 0 0 0 0
plate at 30 °C for 18-24 hr prior to inoculation onto the nutrient
agar. Several colonies of similar morphology of the clinical fungi Gentamycin 10 UI 0 0 15 14
were transferred into Api suspension medium (Biomérieux, Marcy
l’Etoile, France) and adjusted to 2 McFarland turbidity standard In this investigation, Axinella damicornis inhibited
with a densimat (Biomérieux, Marcy l’Etoile, France). The inocula Enterococcus feacalis and Listeria Monocytogenes
of the respective fungi were streaked on to Sabouraud considerably; this indicates that marine sponges
chlorenphenical agar using a sterile swab and then dried. A
sterile filter disc, diameter 6 mm (Whatman paper No. 3) was
remain an interesting source of new antibacterial
placed. 10 µL of extract was dropped on each paper disc (10 metabolites with better activity then some antibiotics.
mg/disc). The treated Petri dishes were placed at 4°C for 1-2 hr This is not surprising because the sponges belonging
and then incubated at 37°C for 18-24 hr. The antifungal activity of to this genus possess a wide variety of compounds
sponge extracts was evaluated by measuring the zone of growth
with different biological activities. Thus during the
inhibition around the discs compared to that produced by 20 µg of
the standard antifungal drug Fongisone. Each experiment was identification of four bioactive substances from
carried out in triplicate and the mean diameter of the inhibition Mediterranean sponges, particularly the organic
zone was recorded. extract of Axinella and Agelas, the sponge extract
RESULTS AND DISCUSSION contains a complex mixture of structurally different
Aqueous extracts from the seven sponges shown a brominated pyrrole alkaloids. Most of these alkaloids
very weak antibacterial activity compared to the ethyl are previously described compounds and were easily
acetate extracts (data not shown) contrary to the identified as 4-bromopyrrole-2-carboxylic acid ,
observation made by Noel Monks et al., (2002). Purealidin A , stevensine , longamide B, and
These authors reported bacterial activity (11 mm clathramides C and D ; zooanemonine ,
halo) attributed to the aqueous extract from Axinella manzacidins A and C, N-metilmanzacidin ,
mukanadins A–C , dibromocantharellin , odiline Considering the high biodiversity of the marine
, 4,5-dibromopalau-amine [20,21], and spongia- sponges together with their phylogenetic age of more
cidin A-D . It is therefore expected that the activity than 600 million years  one question arises: Why
found in the extract of Axinella damicornis might be were these organisms so successful during evolution
due to Axinellamines  or to a vast number of and able to avoid extinction?
alkaloids characteristic of this genus [16,26,27].
Reasons might be found in the fact that sponges
Table II: Antifungal activity of the marine sponge ethyl acetate extracts. have developed an amazingly efficient immune
system reminiscent of that found in vertebrates ;
Fungal growth inhibition diameter (mm)
Marine in addition, sponges seems to have strategies to
C C C C C C
Sponges albicans krusei tropicalis parapsilosis glabrata dubliniensis defend themselves against foreign organisms by
A. Oroides 9 7 0 9 8 0 production of secondary metabolites.
A damicornis 0 0 7 0 0 0
The development of resistance to current antibacterial
P ficiformis 0 0 0 7 7 0 continues to be a serious difficulty in the treatment of
I. sarcotragus 0 0 0 0 0 0 infectious diseases, and therefore the discovery and
I spinosula 0 0 0 0 0 0 development of new antibiotics has become a high
H. communis 0 0 0 0 0 0 priority in biomedical research . In addition, the
Spongia sp. 0 0 0 0 0 0 frequency of invasive fungal infection has risen
Spongia sp. 0 0 0 0 0 0 substantially with increasing numbers of immune-
compromised patients, such as those infected with
10 7 9 8 11 11 HIV, receiving cancer chemotherapy, immune-
suppressive therapy, or treatment with broad-
The antifungal activity tests show that ethyl acetate spectrum antibiotics .
extracts of the seven sponges do not present as CONCLUSION
interesting an activity as that detected for the Sponges collected from the Tunisian Mediterranean
antibacterial test. Indeed, only three among seven coast (Monastir) have been shown to possess a
extracts (Table II) showed capacity of growth specific antibiotic from certain fractions. The most
inhibition of the fungi strains tested. Agelas oroides, interesting species are Agelas oroides and Axinella
which had shown a considerable activity with respect damicornis. These observations corroborate the
to the bacterial strains, presented the most important importance of both genera Agela and Axinella as a
activity against the fungi strains, while Axinella potential source for biologically active compounds
damiconis and Petrosia ficiformis showed very limited such as antibacterial [30,31], antiviral  and
activity. Our results are in accord with observations antitumoral  substances.
made by other authors  who reported significant
antibacterial activities in petroleum ether and acetate To the best of our knowledge, this is the first report
fractions of Ircinia.sp but there was no activity against demonstrating antibacterial and antifungal activity of
fungal strains tested. these species of Porifera. These species are currently
undergoing detailed investigation with the objective of
However, this should be confirmed by using different isolating and identifying the molecular species
assays, e.g., ergosterol inhibition, UV–visible spectra responsible for the activities demonstrated in this
of active sponge extracts and (or) Spheroblast report. Furthermore, the encouraging biological
regeneration . On the other hand, it will be activities seen in this study show that the Tunisian
important to do cytotoxic screening assays for coastline is a potential source of sponge species
potential use of these extracts as antibacterial agents worthy of further investigation.
against human pathogenic bacteria and fungi.
Here, we have shown that a number of sponge We thank the diving centre Alyssa for assistance during
species have activity in in vitro model systems which sponge collection, Professor Abderrahman Bouraoui,
are directly relevant to human disease. Studies of this department of Pharmacology-Faculté de pharmacie de
nature highlight the potential of marine product Monastir for their support during the experiments.
screening programs through which new drugs could Acknowledgements to Dr Karim ben Mustapha, Institut
be identified from the vast, untapped resources that National des Sciences et Technologie de la mer
are within the seas. Salamboo-Tunis for assistance during sponge
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