Ulva intestinalis Extracts and Associated
Bacteria: Biological Potential. Bioactive
Stappia sp. and Extracts from Green Alga
Amel Ismail1,2*, Leila Ktari1, Mehboob Ahmed2,3, Issam Hmila4, Wafa Cherif1, Radhia Mraouna1, Imen Hmani1, Henk Bolhuis2, Lucas J Stal2,5, Abdellatif Boudabous6 and Monia EL Bour1
1Department of Blue Biotechnology & Aquatic Bioproducts -B3Aqua, National Institute of Marine Sciences and Technologies (INSTM), Tunisia
2Department of Marine Microbiology and Biogeochemistry, Royal Netherlands Institute for Sea Research and Utrecht University, The Netherlands
3Department of Microbiology and Molecular Genetics, University of the Punjab, Pakistan
4Laboratory of Epidemiology and Veterinary Microbiology, Pasteur Institute, Tunisia
5Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Netherlands
6Faculty of Mathematical, Physical and Natural Sciences of Tunis, Tunisia
Submission: February 17, 2021;Published: March 08, 2021
*Corresponding author: Amel Ismail, Department of Blue Biotechnology & Aquatic Bioproducts -B3Aqua, National Institute of Marine Sciences and Technologies (INSTM), Tunisia
How to cite this article: Amel I, Leila K, Mehboob A, Issam H, Wafa C, et al. Ulva intestinalis Extracts and Associated Bacteria: Biological Potential. Bioactive Stappia sp. and Extracts from Green Alga. Int J Environ Sci Nat Res. 2021; 27(3): 556213. DOI:10.19080/IJESNR.2021.27.556213
Marine macroalgae and their epibionts represent a promising antimicrobial source. The present work evaluates antimicrobial activities of the green alga Ulva intestinalis Linnaeas (Ulvales, Ulvaceae) crude extracts as well as their surface associated bacteria. U. intestinalis was collected seasonally from the rocky coast of Cap Zebib (Northern Tunisia). Seaweed extracts were prepared by maceration in dichloromethane or dichloromethane/methanol and tested for possible antimicrobial activity against 19 bacteria and fungi. Microdilution test was used to determine Minimal Inhibitory Concentration (MIC). Also, the alga-surface associated bacteria were identified by sequencing the gene coding for 16S rRNA. The bacterial isolates were tested for potential antimicrobial activity by the drop method. U. intestinalis extracts showed a clear seasonal variability of their antimicrobial activity. The lowest MIC was recorded for the winter algae extract with 0.8, 1.6 and 3.2mg/mL against Staphylococcus aureus, Micrococcus sp. and Candida albicans, respectively. In addition, among the epiphytes 2 isolates (U1 and U5) that are closely related to Stappia sp. showed pronounced activities against six microorganisms, notably against Aeromonas salmonicida and Staphylococcus aureus.
Keywords: Epiphytic bacteria; Bioactivity; Antibacterial activity; Green alga
Abbreviations: BSA: Bovine Serum Albumin; D: Dichloromethane; D/M: Dichloromethane/Methanol; DMSO: Dimethylsulfoxide; EDTA: Ethylenediaminetetraacetic; MA : Marine Agar; MB : Marine Broth; MIC : Minimal Inhibitory Concentration; NCBI:
National Center for Biotechnology Information; PCR: Polymerase Chain Reaction; TAE: Tris, Acetate, EDTA; TSA: Tryptic Soy Agar; TSB: Tryptic Soy Broth; CFU: Colony Forming Units
Seaweeds contain various compounds with biotechnological applications . Seaweeds are also widely used as food for human consumption . For many centuries there has been a traditional use of seaweeds as food in Japan, China and the Republic of Korea. Nowadays there are several other countries where the consumption of seaweed is common . Moreover, seaweeds serve as ingredients for food and cosmetics industries and are
also used as fertilizer and as animal feed additive . Besides, seaweeds have several pharmacological applications [4-7].
As a response to environmental pressure many marine organisms and in particular seaweeds produce secondary metabolites. A large number of biologically active compounds with algicidal , antitumor , antifungal , antimicrobial [11,12]and cytotoxic properties  have been attributed to seaweeds. As the result of their photosynthetic activity, seaweeds produce large
amounts of dissolved organic carbon which is released, providing
a highly nutritious habitat for epiphytic microorganisms. Theses
epiphytes protect their host from the surrounding environment
negative effects [14-16]. Moreover, the surface of healthy seaweed
is protected from fouling despite the presence of biofilms of
epiphytic bacteria . This suggests a protective role of epiphytic
bacteria , which helps to maintain the health of algae through
the synthesis of antifouling compounds. These compounds may
have various industrial and medical applications .
Earlier studies focused on the enumeration of the total
seaweed microbial diversity and on the distribution of these
seaweed microbiomes. However, the culturable epiphytic
bacteria associated with macroalgae have been understudied.
A few studies have been conducted on marine algae focusing
on their anti-inflammatory, analgesic, anti-proliferative activity
, antimicrobial activity [20,21], antifouling activity 
and antioxidant activity . In this study, we investigated the
antimicrobial potential of the green alga Ulva intestinalis Linnaeas
(Ulvales, Ulvaceae) collected seasonally from the Northern
Tunisian coast as well as of the culturable epiphytic bacteria of
Ulva intestinalis was collected by hand in shallow water (2m
depth or less) at low tide during winter, spring and summer from
July 2006 to June 2007 from the rocky coast of Cap Zebib (37°16.2
ʹN, 10°3.6ʹE) in the region of Bizerte (Northern coast of Tunisia)
(Figure 1). Samples were kept on ice under aseptic conditions to
prevent contamination and were transferred to the laboratory for
Antimicrobial activity of seaweed extracts and epiphytic
bacteria were tested against a group of pathogenic strains:
Gram-positive (Streptococcus sp. (Pasteur Institute, Tunis),
Staphylococcus aureus (Pasteur Institute, Tunis), S. aureus ATCC
25923, S. aureus ATCC 6538, Enterococcus faecalis ATCC 29212,
Micrococcus sp. (Pasteur Institute, Tunis) and Gram-negative
(Escherichia coli O126-B16 (ATTC 14948), E. coli ATCC 25922, E. coli
ATCC 8739, Vibrio tapetis CECT4600 (Department of Microbiology
and Parasitology, University of Santiago de Compostela, Spain),
V. anguillarum ATCC 12964T, V. alginoliticus ATCC 17749T,
Pseudomonas cepacia (INSTM, Tunisia), P. fluorescens AH2
(Danish Institute for Fisheries Research, Denmark), P. aeruginosa
ATCC 27853, Aeromonas salmonicida LMG3780, A. hydrophila
B3 (RVAU-Denmark), Salmonella typhimurium C52 (Laboratoire
Hydrobiologie Marine et Continentale, Université de Montpellier
II, France) and the yeast Candida albicans ATCC 10231. To
determine the potential sensitivity of the isolates to the host, the
inhibitory activity of the algal extracts was evaluated against its
surface associated bacteria.
After algae sample collection, healthy and thalli with few
epiphytes were selected. The algal thalli were washed three times
rigorously with seawater. Between the washings the water was
removed from the thallus by shaking by hand. Finally, the thalli
were washed with distilled water. Subsequently, algae were dried for 15 days under ambient conditions in the shade or in an oven at
40°C, after which the dry biomass was crushed until a powder was
obtained, which was kept at -20°C until later analysis. Samples from
each collection (fresh seaweed) were kept in 2% formaldehyde.
To prepare the crude extract of the algae, 20g of the dried algal
biomass was extracted successively by 2 organic solvents of
increasing polarity, dichloromethane (D) and dichloromethane/
methanol (D/M) (1:1 v/v). These solvents are suitable to recover
non-polar and moderately polar algae compounds.
Each extraction (24h at room temperature) was repeated 3
times. The extracts were pooled and filtered and the filtrate was
concentrated in a rotary evaporator. The crude extract was stored
at -20°C until use.
Epiphytic bacteria were isolated from the surface of U.
intestinalis collected during the winter and during the summer.
After collection of the algae and upon arrival at the laboratory, the
algae were washed 3 times with autoclaved seawater (brought
from the sample site) in order to remove loosely attached
bacteria . Then, 10g of algal biomass was agitated on a shaker
(Stomacher 400 circulator) in 90mL of autoclaved seawater for
6min. This homogenate (1mL) was diluted (serial dilutions of
1/10) with autoclaved normal saline (0.9% NaCl solution in
distilled water) until 10-3. 100μL of each dilution (10-1, 10-2 and 10-
3) was plated on marine agar 2216 (MA) (Pronadisa laboratories,
CONDA). After incubation for 7 days at 20°C  visible bacterial
colonies were selected. The bacteria cultures were purified by
repeatedly streaking on MA until pure cultures were obtained.
Pure cultures were stored at -80°C in marine broth 2216 (MB)
(Pronadisa Laboratories, CONDA) supplemented with 20% sterile
The bacterial colonies growing on agar were suspended
in sterile Milli-Q water and used as template for PCR reactions.
The bacterial 16S rRNA gene was amplified using two universal
primers B8F and U1492R  (Table 1). PCR reactions were
performed using a DNA Thermal Cycler Gene Biometra T1
(Perkin-Elmer Co. Norwalk CT, USA) in 25μL final reaction volume
containing 0.1μL hotstart DNA polymerase (Qiagen), each primer
at a final concentration of 10pmol μL-1, each desoxynucleoside
triphosphate at a concentration of 200μM, 1.25μL DMSO
(dimethylsulfoxide), 2.5μL BSA (Bovine Serum Albumin) at 0.2mg
mL-1 final concentration, 2.5μL PCR buffer (containing MgCl2) and
1μL of DNA template. The PCR protocol consisted of 40 cycles
of denaturation at 94°C for 30s, annealing at 55°C for 30s and
extension at 72°C for 1min 50 . The cycles were preceded by 15
min of denaturation at 94°C and ended with a final extension of 7
min at 72°C. Negative controls contained the PCR reaction mixture
without template DNA.
PCR products (2μL) were analyzed by electrophoresis on
agarose gel (1% w/v agarose, 1X TAE running buffer containing
40mM Tris-acetate and 1mM EDTA, pH 8). Electrophoresis was
performed at 100V for 45min. The gels were stained for 45min
with SYBR Gold (Invitrogen Corp.) and visualized under UV.
DNA sequencing was done using the BigDye Terminator (Big
Dye Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems)
according to the manufacturer’s instructions (Using 100ng of the
PCR product). The sequencing reactions were performed on an
ABI 377 DNA sequencer (Applied Biosystems) using primers C5
, C26 , C72  and C112  (Table 1).
Sequences of the 16S rRNA gene were compared with public
nucleotide sequences using alignment tools local database:
BLAST (http://www.ncbi nlm.nih.gov/BLAST), Research Program
GenBank National Center for Biotechnology Information (NCBI).
This database allows alignment based on the primary and
secondary structure of the 16S rRNA. Phylogenetic analysis of
the sequences of these isolates and related species was carried
out using the neighbor-joining (NJ) method, which is based on
distances notion using MEGA version 4.1 software algorithm .
16S rRNA genes partial sequences of the U. intestinalis
epiphytic bacterial community (10 sequences) have been
submitted to NCBI - GenBank and had the following accession
numbers: (U1-U10) (FN821679-FN821688).
Bacterial isolates were tested for their antimicrobial activity
against bacteria and the yeast (C. albicans) by adopting the spot
method which consists in depositing spots of 20μL of bacterial
suspension (culture in trypto-casein-soy broth (TSB, BIORAD)
containing 20g/L NaCl) of each isolate, on trypto-casein-soy agar plates (TSA, BIORAD) containing 20g/L NaCl already seeded
with a confluent layer of the indicator strain (dried at 30°C for
30min). Simultaneously, a spot of 20μL of only TSB medium is
deposed as negative control. The plates were then incubated at
30°C for 24h as described by Rao et al. (2005) . Antimicrobial
test of U. intestinalis extracts against its isolates was done by the
disc’s diffusion method (A qualitative method used only for the
detection of the presence of eventual activity): 500μg of algal crude
extract was dissolved in dichloromethane or dichloromethane/
methanol (10μL) and placed on sterile filter paper discs (6mm).
After solvent evaporation, the discs were placed on TSA plates,
already inoculated with a test culture (106 bacteria. mL-1) in
TSB. Simultaneously, a disc loaded with only the solvent was
used as a negative control. Plates were incubated overnight
at 30°C. Inhibition diameters (mm) were measured after 24h.
Antimicrobial activity tests were conducted in triplicate.
To determine the MIC for selective active extracts, the TSB
media was used. Essays were performed in sterile culture plates
of 96 round bottom wells. Suspensions of indicator bacterial
inoculum were adjusted in the sterile broth medium TSB to
correspond to the density of about 0.5 Standard McFarland
(Corresponding to 0.063 optical density at 600nm, approximately
108 CFU mL-1), then diluted 10-fold twice to obtain a bacterial
suspension density of about 106 CFU mL-1. Microplates wells were
inoculated with 180 μL of the culture containing the inoculum.
20μL of each concentration of seaweed extract (diluted in
dimethyl sulfoxide (DMSO)) were added to the wells containing
the bacterial culture suspension. The negative control contained
200μL of the culture medium only (without alga extract). Extracts
previously solubilized in the DMSO (20μL) were adjusted to give
a concentration range of 3200 to 50μg/mL Tests were performed
in duplicate and plates were incubated for 18-24h in 37°C.
Subsequently, the wells were examined for bacterial growth as
indicated by turbidity. The last well in the dilution series that
did not show growth correspond to the MIC of the antimicrobial
Statistical analysis of the data was carried out using SPSS
personal computer statistical package (version 20, SPSS Inc,
Chicago). Variation in different treatments was measured in terms
of standard error whereas significant differences among different
treatments were determined by applying ANOVA (p = 0.05) and
further Duncan’s Multiple Range Test as post-hoc test.
Crude extracts of the algae collected in winter were the most
active and showed the largest antimicrobial activity against
indicator microorganisms tested. Of the 19 species tested, 11 (10
bacteria and the yeast C. albicans) were sensitive to extracts of the
algae collected in winter. Highly significant activity was detected
for the dichloromethane extracts, which were active against S.
aureus, Micrococcus sp. and C. albicans strains (Table 2). Seaweed
extracts of algae collected during spring were weakly active
against the 3 strains of S. aureus and against Micrococcus sp. No
antibacterial activity was detected in extracts of the algae collected
during summer, while in autumn algae were totally absent at
the collection site. The effect of the solvent and of the season of
collection on the algal extract’s antibacterial activity against S.
aureus ATCC 6538 is shown in Figure 2. Dichloromethane extracts
of the algae collected in winter were the most effective. MIC of
winter U. intestinalis dichloromethane and/or dichloromethane/
methanol crude extracts were determined against S. aureus ATCC
25923, S. aureus ATCC 6538, Micrococcus sp. and C. albicans as
illustrated in Table 3. MIC values of dichloromethane extracts were
50% lower than those of D/M extracts and they range from 0.8-
1.6mg/mL and 1.6-3.2mg/mL, respectively. Among the indicator
species, the 3 cultures of E. coli, Vibrio sp., S. typhimurium and E.
feacalis were resistant to all seaweed extracts.
W: winter; Sp: spring; Su: summer; (-): no activity; D: dichloromethane; D/M; dichloromethane/methanol
Mean value of 3 replicates ± standard error values. The different letters indicate significant differences between inhibition zone by different strains
using Duncan’s Multiple Range Test (DMRT) at P = 0.05.
D: dichloromethane; D/M: dichloromethane/methanol.
Mean value of 3 replicates ± standard error values. The different letters indicate significant differences between inhibition zone by different strains
using Duncan’s Multiple Range Test (DMRT) at P = 0.05.
During this study a total of 17 morphologically distinct
bacterial strains (U1-U17) was isolated from the surface of U.
intestinalis collected in winter and summer. The majority of
isolates was obtained from the surface of the algae collected in
winter (11 isolates coded U1, U3-U7, U9- U11, U13 and U14). The
number of isolates from algae collected in summer was lower (6
isolates coded U2, U8, U12, U15-U17). All 17 isolates were tested
for their potential antibacterial and antifungal activities against
the 19 indicators microorganisms. Amongst the 17 isolates
only 10 (U1-U10) could be identified. Thus, by 16S rRNA gene
amplification and sequencing we identified 2 groups: Gram (+)
and Gram (-). The Gram (-) group consisted of Proteobacteria
with 1 isolate belonging to Gammaproteobacteria (Vibrio sp.), 7
isolates belonging to Alphaproteobacteria (Genera Stappia, Nisaea,
Ruegeria, Sphingopyxis, and Loktanella) and 1 Bacteroidetes (one
isolate closely related to genus Lewinella). The Gram+ isolates
belonged to a single group: Firmicutes (Genus Planomicrobium)
(Table 4). The results suggest the presence of a diverse microbial
community associated with U. intestinalis with a Simpson index D
of 0.06. The phylogenetic tree constructed based on the 16S rRNA
gene sequences of U. intestinalis isolates and similar sequences in
Genbank are shown in Figure 3.
Amongst the 17 U. intestinalis isolates, two (U1 and U5)
showed antibacterial activity. These two strains were identified
as Stappia sp. with an identity of 99 and 98% for U1 and U5,
respectively. These strains showed antibacterial activity against 6
indicator species: S. aureus ATCC 25923, S. aureus, A. salmonicida,
Streptococcus sp., P. aeruginosa and A. hydrophila. The strongest
inhibitory activity was from U5, which acts against S. aureus and
A. salmonicida with inhibition halos of 13 and 12mm, respectively
(Table 5, Figure 4). To verify whether the U. intestinalis epiphytic
bacteria were resistant and specific to their host; the 6 algal
extracts (D and D/M extracts of the alga collected in winter,
spring, and summer) were tested for their inhibitory effect against
the 17 associated bacteria. The results show that all U. intestinalis
epiphytes were resistant to the algal extracts, except isolate U8,
which is closely related to Vibrio sp. A significant difference was
found between the number of indicator bacteria inhibited by
the U. intestinalis extracts (10 out of 18 bacteria tested (= 55%),
(Table 2) and the number of epiphytic bacteria inhibited by the
same extracts (1 out of 17 isolates (= 5.8%)) (Table 5).
In order to elucidate the origin of the antimicrobial
compounds, the activity of the algal extracts was compared with
that of the epiphytic bacteria (Table 6). It was shown that some of
the indicator strains (A. hydrophila, A. salmonicida, P. aeruginosa
ATCC 27853, S. aureus ATCC 25923, S. aureus and Streptococcus
sp.) were sensitive to both the algal extracts as well as to the
associated isolates (U1 and U5). Other pathogens such as P.
fluorescens AH2, P. cepacia, S. aureus ATCC 6538, Micrococcus sp.
and C. albicans were only sensitive to the algal extracts.
Inhibition zone (mm) measured by considering the diameter of the spot,
(0): no activity. Mean value of 3 replicates ± standard error values. The
different letters indicate significant differences between inhibition zone
by different strains using Duncan’s Multiple Range Test (DMRT) at P =
The seasonal variability of U. intestinalis inhibitory activity
against a number of indicator species was tested. The seasonal
variation of seaweed toxicity has previously been described
[20,32-34]. Also, in this study the strains S. aureus, Micrococcus
sp. and C. albicans were strongly inhibited by the dichloromethane
extracts of U. intestinalis that were collected in winter. The seasonal
variation of inhibitory activity could not be attributed to a single
biological process. Seasonal peak of activity has been associated
to physiological phenomena  as the rate and the process of
photosynthesis. Biotic and abiotic environmental factors may
also affect the seasonal variation of antibacterial . The peak
of bioactivity observed in our study may also be related to the
reproductive or growth period, as was shown for green, brown
and red alga [33,35]. However, in other cases, such as for the red
alga Osmundea truncata, the peak of bioactivity occurred after the
reproductive period . Marti et al.  stated that bioactivity
peak may be related to processes of ageing and allocation of
resources from growth or reproduction to production of toxic
Comparing our findings with those reported in other studies,
we hypothesize that geographic location has an impact on the
antimicrobial activity observed for U. intestinalis. In a study carried
out by Ktari et al. , the D and D/M extracts of U. intestinalis
collected during winter from the Tunisian coast (although the
collection site was different: the coast of Monastir), was inactive
when tested for its antibacterial activity against S. aureus. This
finding disagreed with what we report here. This pathogen was
the most sensitive of all for the extracts of this alga collected
in winter and extracted by the same procedure and solvents.
Berber et al.  studied antimicrobial activity of U. intestinalis collected in winter from the coastal region of Sinop, Turkey and
reported that the alga was inactive against Micrococcus and S.
aureus 25923, the latter was the same reference strain used in
our study (and was the most sensitive strain). Also, in this study
U. intestinalis collected in spring had moderate inhibitory activity
against S. aureus while the same alga species collected in spring
from the coast of Pakistan did not show any activity against this
These results and observations support the hypothesis of the
geographical location impact on the algae secondary metabolites
production. This variation related to collection site might be due
to the nature of the site. Whether it is exposed to strong shear
forces or to calm conditions, in the open sea or in protected bays.,
different biotic and abiotic environmental factors will impact the
biology and physiology of these seaweeds, and hence influence
the production of secondary metabolites. Marti et al.  pointed
out that also various ecological parameters such as nutrients
and irradiance may determine the production of secondary
Several studies noted antimicrobial activity of U. intestinalis
against various bacteria including B. subtilis, S. aureus, E. faecalis,
P. aeruginosa, V. fluvialis, E. coli, S. pneumoniae, P. mirabilis and
V. cholera [38-41]. Our results show inhibitory activity of U.
intestinalis extracts against 10 different bacterium strains as
well as against the yeast C. albicans. Dichloromethane extract of
U. intestinalis seem to be more effective against bacteria than the
yeast as we report a relatively low MIC of 0.8mg/mL for S. aureus
and 3.2mg/mL for the yeast. In a study by Klongklaew et al. 
the MICs determined for U. intestinalis crude extract against Vibrio
spp., ranged between 5 and 10mg/mL. Therefore, the active
compound against S. aureus seems to be present in considerable
quantity in the nonpolar extract. Finding new natural bioactive
compounds is needed because of the increasing number of
antibiotic-resistant pathogens, particularly in nosocomial
infections. Therefore, it was interesting to observe the inhibitory
activity of U. intestinalis against S. aureus, a causative agent of toxic
shock syndrome, which can affect nearly every organ of the body,
especially in the case of hospitalized people.
Previously, representatives of the genus Stappia have been
isolated from the marine environment: seawater [43,44], oysters
, dinoflagellates , cyanobacterial communities 
and certain marine macroalgae . Chen et al.  reported
an algicidal effect of Stappia sp. while Zhu et al.  showed
antibacterial and cytotoxic activity of Stappia aggregata isolated
from a dinoflagellate. Antibacterial effects of U. intestinalis towards
strains of Pseudomonas and Alteromonas have also previously
been reported .
In this study we report the isolation of epiphytic bacteria
from the surface of U. intestinalis that belong to the genus Stappia
(strains U1 and U5), which possess strong inhibitory activity
against the pathogens S. aureus and A. salmonicida (U5) and a
moderate inhibition against A. salmonicida, Streptococcus sp.,
P. aeruginosa and A. hydrophila (U1). Lemos et al.  reported
that 17% of the epiphytic bacteria of marine algae (mainly U.
intestinalis) show antibacterial activity against the pathogen S.
aureus. Therefore, it can be concluded that bacteria isolated from
U. intestinalis are a potential source of secondary metabolites that
act against this pathogen.
With regard to the observed seasonal variation of the
antibacterial activity of the algae, the two Stappia sp. isolates
(U1 and U5) may be responsible for an important part of the
antibacterial activity of extracts of U. intestinalis collected in
winter. The isolates U1 and U5 were isolated from the alga during
winter. Another isolate of the same genus Stappia (isolate U2) that
was isolated in summer did not show any activity against indicator
bacteria. These observations suggest that bacteria of the same
genus and isolated from the same species of alga may produce
different secondary metabolites according to the season. This can
be explained by the variation of the temperature, the pH, fouling
organisms on the host surface and a multitude of other abiotic and
biotic factors that may be involved in controlling and influencing
secondary metabolite production by epiphytic bacteria.
It is known that in the marine environment mutual
interactions between epiphytic bacteria and their host generate
a typical bacterial community that is tightly associated with
the host . The majority of algae seem to require a bacterial
community on their surfaces in order to grow properly .
Algae may develop defense mechanisms against exogenous
pathogens and against certain unwanted bacteria that colonize
their surfaces (biofouling) . Such defense mechanisms may
include the production of antibiotic substances that prevent
attachment of fouling microorganisms. Hence, by testing the
effect of secondary metabolites secreted by algae (U. intestinalis
extracts) the degree of affinity of epiphytic bacteria to their host
can be assessed. Isolates that show resistance to the algal host
extract may be considered specific for their host and might be
beneficial. This was the case for 16 isolates U1-U7 and U9-U17.
Whereas isolates showing sensitivity to the algal host extract are
considered to be non-specific or potentially pathogenic. The host
attempts to refuse such bacteria as was the case for Vibrio strain
U8. Except strain U8, in this study, U. intestinalis epiphytes were
resistant to their host extracts. The inhibition of Vibrio species
colonizing the surface of U. intestinalis is important since Vibrio
is known to be involved in the mineralization of organic material
in the sea and for causing disease [52,53]. Vibrio species are
opportunistic pathogens on several diseased algae thalli .
Thus, Vibrio might have had a negative impact on U. intestinalis
health and would explain its inhibition by the host’s secondary metabolites. All other algae isolates were resistant against their
host extracts. This suggests that U. intestinalis epiphytes are well
adapted to their environment and the antibacterial substances
produced by their host. Epiphytic bacteria living on U. intestinalis
surface may help their host by contributing to the production of
bioactive compounds, which would prevent colonization of the
algal surface by other, potentially harmful bacteria. Several pilot
studies demonstrated that some bacteria promote algal growth,
whereas others induce their morphogenesis. This is particularly
the case of the order Ulvales [55-57].
Several indicator pathogens tested in this study appeared to
be sensitive to both the algal extracts as well as to the associated
isolates (U1 and U5). These two active isolates alone may be
responsible for the observed inhibitory activity or, alternatively,
involved in the production of bioactive compounds. This is also
supported by the fact that these strains were isolated from algae
collected in winter when antibacterial activity peaks. Other
pathogens such as P. fluorescens AH2, P. cepacia, S. aureus ATCC
6538, Micrococcus sp. and C. albicans were only sensitive to the
algal extracts. It is therefore concluded that the algae produces
bioactive secondary metabolites for self-defense [58,59].
U. intestinalis expresses antimicrobial properties more
pronounced during the winter. Because these algal toxins are
selective, several epiphytic bacteria can live in close association
with the host while preventing pathogenic bacteria from colonizing
the alga. U. intestinalis hosts a diverse community of culturable
bacteria that produce bioactive compounds able to inhibit Gram
(+) and Gram (-) bacteria as well as the yeast C. albicans. Among
the epiphytic bacteria, isolates closely related to the genus Stappia
showed particularly antibacterial activity. Given the fact that it
is easy to cultivate these epiphytic bacteria producing bioactive
compounds, these bacteria may be of considerable interest for
the pharmaceutical industry. This would allow the exploitation
of seaweed as a natural source of antimicrobial compounds while
preserving its natural stock.
The authors thank Ms. Veronique Confurius-Guns, Department
of Marine Microbiology and Biogeochemistry, Royal Netherlands
Institute for Sea Research and Utrecht University (NIOZ), for her
assistance and help in the identification of bacterial isolates.