This study aimed to identify natural anti-fouling compounds from marine bacteria. Twenty-four bacteria strains isolated from marine sponges were tested for antibacterial activity against 10 target bacteria strains. Eleven bacteria strains maintained their antibacterial activity. Eight of these were selected for further analysis. Extracts from the bacteria and their growth media were tested against the target bacteria, with some extracts showing activity against multiple targets. The extracts were also tested against four environmental fungi strains, with some demonstrating good anti-fungal potential. One bacteria strain, Streptomyces sp. ES3, was chosen for further compound isolation due to its broad-spectrum antibacterial activity.

1. RESEARCH POSTER PRESENTATION DESIGN © 2012
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Bio-fouling is a natural process occurring in the marine environment. One of the initial
steps in fouling succession is the adhesion of microorganisms to the surface. Natural
compounds with antimicrobial properties have an anti-fouling potential while being
environmentally friendly. The aim of this study was to find such anti-fouling
compounds.
INTRODUCTION
MATERIALS & METHODS
CONCLUTIONS
Eight extracts were checked against 4 environmental fungi previously isolated in our
lab: Aspergillus (NF10) and Penicillium (NF1) isolated from seawater in the
Mediterranean sea from different depths, and Trichoderma (NF14) and Penicillium
(NF27) isolated from Mediterranean sediment at different depths. The growth of fungi
was determined by measuring absorbance at 620nm in 96 well plate and calculating
the percentage of growth in crude extracts compared to controls (Table 3).
•Not all of the bacteria maintained their activity after being frozen for a few years.
•The ES3 strain was chosen for further isolation of natural products because of its
anti-bacterial properties.
•Anti-fungal assays revealed a good potential for having activity in extracts previously
found as non-active in anti-bacterial disc diffusion assay.
ACKNOWLEDGEMENTS
Overall 30 extracts have been made of bacteria and their media. Out of these, nine
were active in disc diffusion assay (Table 2, Figure 4), among them four extracts
were active against at least five target bacteria. The source of these active extracts
includes Streptomyces sp. (strains ES3 and ES4) isolated from A. erina and the
extracts of the agar were found to be more active than the bacterial extracts.
24 bacteria strains (Figure 1) previously isolated in our lab mostly from sponges of
the genus Amphimedon and two from Axinella polypoides were found to be active in
anti-bacterial streak assay and were chosen for this work.
The bacteria were grown from stocks (-80ᵒC), identified morphologically and
molecularly, and checked in anti-bacterial streak assay in order to confirm their
activity against 5 Gram-positive and 5 Gram-negative bacteria (4 of which are
laboratory strains and 6 environmental bacteria isolated from seawater and sediment
in the Mediterranean sea).
The bacteria which preserved their anti-bacterial activity were grown on several
plates with the medium on which they were active previously (ISP2), in different
conditions (in liquid, on agar; shaken or stationary; different growth periods) (Figure
2). The medium was extracted in ethyl acetate and bacteria in methanol. The crude
extracts were tested by applying disc diffusion assay against target bacteria. Eight
random extracts were tested against environmental fungi, as well.
1 – Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 6997801, Israel
2 – School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 6997801, Israel
*fayana3@gmail.com
Yana Farovich1*, Shmuel Carmely2, Micha Ilan1
Anti-microbial compounds from sponge associated bacteria
RESULTS
11 out of 24 bacteria maintained their anti-bacterial activity in streak assay
(Table 1, Figure 3). Out of these, eight bacteria were active against at least four
target bacteria and were therefore chosen for further natural products isolation.
Table 1: Mean distance of inhibition by bacteria of interest in anti-bacterial streak
assay (after 48 h)
Gram (+) positive; Gram (-) negative; (-) no available data
* After 48 h the NB90, ESY10 & ESY9 bacteria showed no visible growth, so they were checked also after
72 h.
Table 1 does not contain data about Pseudomonas aeruginosa (lab strain) and Pseudomonas sp. (NB86) environmental
bacteria as all the checked bacteria were non-active against them and Escherichia coli GM 1655 (lab strain), as only
AA71 yellow was active against it (mean inhibition of 3 mm).
Table 2: Mean zone of inhibition by active crude extracts in disc
diffusion assay (after 48 h)
Gram (+) positive
Table 3: Percentage of growth for environmental fungi in crude
extracts compared to controls (after 72 h)
Activity threshold
was 75% inhibition.
Figure 3. Anti-bacterial streak assay of ES10
bacteria. The numbers 3,4,5 and 6 represent ESY10,
NB90, NB86 and ESY9 accordingly.
Shaken
Stationary
ES10 CI16 AI13 control
Figure 1. Plates representing
diversity of isolated bacteria. Figure 4. Anti-bacterial disc diffusion assay of four
active extracts. The numbers 1, 2, 3 and 4
represent ES3 agar, ES4 agar, ES3 bacteria and ES4
bacteria accordingly; 5 represents control without
extract on ESY9 bacterium.
Staphylococcus
aureus
Staphylococcus
albus
Lysinibacillus
sp. (ESY9)
Kocuria sp.
(ESY10)
Sporosarcina
sp. (NB90)
Vibrio sp.
(ESY12)
Sulfitobacter
sp. (ESY17)
Streptomyces
sp. (ES10)
Amphimedon
erina
8 18 16 14 17 22 0 0
Streptomyces
sp. (ES4)
A. erina 8 8 1 5 4 2 0 0
Streptomyces
sp. (ES3)
A. erina 8 9 4 14 12 10 0 2
* Streptomyces
sp. (CS3)
Amphimedon
compressa
8 2 1 0 12 7 0 0
Streptomyces
sp. (CI16)
A. compressa 8 8 7 0 17 10 0 17
Kocuria sp.
(AA71 yellow)
Amphimedon
chloros
5 3 0 - - - 0 0
Not specified
(AR23)
A. chloros 5 11 9 4 8 10 0 0
N.S. (AI13) A. chloros 8 6 12 9 30 26 0 0
Kocuria sp.
(AM48 dots)
A. chloros 5 0 0 0 7 6 - -
Actinomycetales
order (MPII.15)
Axinella
polypoides
8 - - 16 32 24 9 0
Streptomyces
sp. (MPII.N1)
A. polypoides 8 - - 13 29 14 4 0
Environmental bacteria
Mean distance of inhibition (mm)Bacterial
genus/ order
(code)
Sponge
origin
Growth
period
(days)
Lab strains
a
Staphylococcus
aureus
Staphylococcus
albus
Lysinibacillus
sp. (ESY9)
Kocuria sp.
(ESY10)
Sporosarcina
sp. (NB90)
Streptomyces
(ES3)
agar 12 17 22 18 15
Streptomyces
(ES3)
bacteria 2 0 13 16 9
Streptomyces
(ES4)
agar 10 17 20 18 13
Streptomyces
(ES4)
bacteria 8 8 16 16 10
Streptomyces
(MPII.N1)
agar 0 10 0 8 0
Streptomyces
(MPII.15)
agar 0 0 9 0 0
Streptomyces
(ES10)
bacteria 0 0 0 7 0
Mean zone of inhibition (mm)Bacterial
genus
(code)
Type of
extract
Code of bacteria and
conditions of liquid
medium/ agar
Penicillium (NF27) (%) Penicillium (NF1) (%) Trichoderma (NF14) (%) Aspergilus (NF10) (%)
AI13 shaken 41.3 81.7 23.8 0.0
AI13 stationary 47.3 78.8 16.5 10.9
ES10 shaken 16.8 22.9 23.6 0.0
ES10 stationary 45.4 67.6 32.9 63.9
CI16 shaken 37.8 59.4 8.1 13.8
CI16 stationary 60.8 71.8 27.4 27.1
MPII.15 agar 28.2 45.4 28.9 36.5
MPII.N1 agar 33.1 61.0 14.1 0.0
This research has received partial funding from the European Union Seventh
Framework Programme (FP7/2007-2013) under grant agreement n° KBBE-2010-266033
(SPECIAL).
Figure 2. An experiment with shaken
and stationary conditions for three
bacteria of interest, in liquid ISP2
medium and control without bacteria.