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High reduction of staphylococcal biofilm by aqueous extract from marine

sponge-isolated Enterobacter sp

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DOI: 10.1016/j.resmic.2020.10.002

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Contents lists available at ScienceDirect

Research in Microbiology
journal homepage: www.elsevier.com/locate/resmic

Original Article

High reduction of staphylococcal biofilm by aqueous extract from

marine sponge-isolated Enterobacter sp.
Suzanne de Oliveira Nunes a, 1, Heloisa da Silva Rosa a, 1, Anna Luiza Bauer Canellas a,
Maria Teresa Villela Romanos a, Katia R.N. dos Santos a, Guilherme Muricy b,
Walter M.R. Oelemann a, Marinella Silva Laport a, *
a
es, Universidade Federal Do Rio de Janeiro, Av. Carlos Chagas Filho, 373, Cidade Universita
Instituto de Microbiologia Paulo de Go
ria, 21941-590, Rio de
Janeiro, RJ, Brazil
b
Museu Nacional, Universidade Federal Do Rio de Janeiro, Quinta da Boa Vista s/no., Sa ~o Cristo ~o, 20940-040, Rio de Janeiro, RJ, Brazil

va

a r t i c l e i n f o a b s t r a c t

Article history: Staphylococcus aureus and Staphylococcus epidermidis are among the most important bacterial species
Received 6 May 2020 responsible for biofilm formation on indwelling medical devices, including orthopaedic implants. The
Accepted 5 October 2020 increasing resistance to antimicrobials, partly attributed to the ability to form biofilms, is a challenge for
Available online xxx
the development of new antimicrobial agents. In this study, the cell-free supernatant obtained from
sponge-associated Enterobacter strain 84.3 culture inhibited biofilm formation (>65%) and dissociated
Keywords:
mature biofilm (>85%) formed by S. aureus and S. epidermidis strains. The culture supernatant was
Antibiofilm agent
subjected to solvent partitioning and the aqueous extract presented a concentration-dependent anti-
Marine enterobacter
Marine sponges
biofilm activity for each strain with a minimum biofilm eradication concentration (MBEC) ranging from
Medical device-associated infections 16 to 256 mg/mL. The effect of the aqueous extract on mature S. aureus biofilm was analyzed by confocal
Staphylococcus aureus scanning laser microscopy, showing a significant reduction of the biofilm layer as well as diminished
Staphylococcus epidermidis interactions among the cells. This extract is not toxic for mammalian cells (L929 cell line). Studies tar-
geting substances with antibiofilm activity gained significant attention in recent years due to difficult-to-
treat biofilm infections. Here, sponge-associated Enterobacter 84.3 proved to be a source of substances
capable of eradicating staphylococcal biofilm, with potential medical use in the future.
© 2020 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

1. Introduction strategy to evade the host’s immune system and is often a source
of bacterial spread to other body sites. Biofilms show an inherent
Staphylococci are currently the major agents of medical resistance to antimicrobials [5,6] and an increased rate of hori-
device-related infections [1,2]. In orthopaedic prosthetic in- zontal genetic transfer leading to the acquisition and spread of
fections, the most commonly Gram-positive cocci isolated are multidrug-resistance [1e3].
Staphylococcus aureus and coagulase-negative staphylococci (CNS) Today there are no therapies that effectively target staphylo-
[3]. S. aureus strains causing implant infections show high rates of coccal biofilms, and none of the known modulators of virulence
antimicrobial resistance, and there is an alarming increase of has been approved yet for clinical use [6]. This indicates the need
antimicrobial resistance observed in other species, such as for new antibiofilm molecules. Marine sponge-associated bacteria
Staphylococcus epidermidis [1,3,4]. Implant infection causing bac- represent a still underexploited source of biodiversity able to
teria generally form biofilms, in which bacterial aggregates tightly synthesize a broad range of bioactive substances, including anti-
adhere to the biomaterial surface and are responsible for the biofilm agents with good performance against clinical isolates
persistence of implant infections. Biofilm serves as a bacterial [7e10].
In a previous study, we built a collection of culturable marine
sponge-associated bacteria isolated from samples of the marine
* Corresponding author. sponge Oscarella spp. from Cabo Frio, SE Brazil [11]. In search of
E-mail addresses: [email protected], [email protected] potential antimicrobial substances, 85 bacterial isolates were
(M.S. Laport). tested against indicator bacteria of medical importance in agar
1
Both authors contributed equally to this work.

https://doi.org/10.1016/j.resmic.2020.10.002
0923-2508/© 2020 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Please cite this article as: S.O. Nunes, H.S. Rosa, A.L.B. Canellas et al., High reduction of staphylococcal biofilm by aqueous extract from marine
sponge-isolated Enterobacter sp., Research in Microbiology, https://doi.org/10.1016/j.resmic.2020.10.002
S.O. Nunes, H.S. Rosa, A.L.B. Canellas et al. Research in Microbiology xxx (xxxx) xxx

diffusion assays, where cells experience quorum sensing. Twenty- 2.3. Antibiofilm activity
seven (31.7%) of them showed inhibitory activity against at least
one clinical indicator strain assayed, including some biofilm 2.3.1. Cell-free supernatant
producers [11]. Aiming to find substances that eradicate biofilm Marine Enterobacter culture supernatant was evaluated for its
rather than bacterial growth, supernatants of the remaining 58 capacity to either inhibit biofilm formation or to disrupt biofilm
strains grown under planktonic conditions were evaluated for previously formed by the reference and clinical strains. Antibiofilm
antimicrobial activity against the reference strains S. aureus ATCC activity was performed as previously described [16] with modifi-
29213, S. aureus ATCC 25923 and S. epidermidis ATCC 35984. The cations. Briefly, in the wells of a 96-well microtiter plate (TPP,
cell-free supernatant from Enterobacter strain 84.3 showed no Trasadingen, Switzerland) 20 mL of the indicator strain culture
inhibitory activity against the staphylococcal strains (data not (~108 CFU/mL) were mixed with either 180 mL of the cell-free su-
shown). Thus, this strain was selected as potential producer of pernatant or 180 mL of BHIg medium (negative control). All tests
antibiofilm agents, because these agents may impose a weaker were carried out in triplicate and the plate was incubated aerobi-
selective pressure for the development of drug resistance relative cally for 24 h at 37  C. After incubation, the wells were washed
to current antibiotics. Besides, antibiofilm substances may be co- three times with phosphate-buffered saline (PBS; pH 7.4). The
administered with antibiotics and some promising results in vitro remaining attached bacteria were fixed by exposure to hot air at
have been reported [12]. Therefore, in this study Enterobacter 60  C for 60 min, and the wells were stained with 0.2% crystal violet
strain 84.3 was screened for the capacity to inhibit biofilm for- for 15 min at room temperature. The plate was air-dried, and the
mation and to dissociate mature biofilm of Staphylococcus spp. dye retained by adherent cells was dissolved in 95% ethanol. The
reference strains as well as clinical strains. optical density of each well was measured at 570 nm using a mi-
crotiter plate reader (model 680, Bio-Rad Laboratories, Hertford-
shire, UK). The inhibition of biofilm formation of each strain was
2. Materials and methods expressed as percentage of biofilm reduction, by applying the
following formula: (ODcontrol - ODsample/ODcontrol)  100. Three in-
2.1. Bacterial strain and culture condition dependent experiments were performed in triplicate and therefore
each data point was averaged from a total of nine values obtained
Sponge-associated Enterobacter strain 84.3 (MK780772.1) was and the standard deviation (SD) was calculated.
isolated from the Brazilian sponge Oscarella spp. as previously Enterobacter 84.3 supernatant was also evaluated for its capacity
described [11]. Cell-free culture supernatants were prepared by to dissociate mature biofilm produced by the reference strains. As
inoculating 107 cells of Enterobacter 84.3 in 3 mL of Brain Heart mentioned above, three independent experiments were carried out
Infusion (BHI) medium (Difco, MI, USA). After incubation at 25  C in triplicate. Twenty microliters of indicator bacteria (~108 CFU/mL)
for 24 h, 22 mL of BHI containing 1% glucose were added and the in 180 mL of BHIg were loaded into wells of a microtiter plate and
culture further incubated at 25  C for 48 h. After centrifugation at incubated at 37  C for 24 h. Planktonic cells were removed by
4000g for 20 min (Eppendorf centrifuge 5804 R, Hamburg, Ger- washing the plate three times with PBS. To the remaining pre-
many) the supernatant was sterilized by filtration (Millipore formed staphylococcal biofilm, 200 mL of the cell-free supernatant
0.22 mm) and kept at 4e7  C until use. were added per well. Control samples of each staphylococcal bio-
Six strains classified as strong biofilm producers were film were kept without the addition of cell-free supernatant. After
included in the study as indicator strains and positive control for another 24 h at 37  C the plates were again washed in PBS, heat-
the antibiofilm assays: three reference strains of the American fixed and stained as described above. The ability to dissociate bio-
Type Culture Collection (ATCC), S. aureus ATCC 29213, S. aureus film was also expressed as antibiofilm activity (%), by applying the
ATCC 25923 and S. epidermidis ATCC 35984; and three clinical above-mentioned formula.
strains isolated from orthopaedic prosthetic infections,
S. aureus1117a and S. aureus11123a, and from surgical site infec-
2.3.2. Extracts
tion, S. epidermidis 24 [13,14]. All Staphylococcus strains were
To determine the antibiofilm activity, the extracts were tested in
grown on BHI agar for 18 h at 37  C or in BHI broth supplemented
serial dilutions ranging from 1024 to 1 mg/mL in BHIg medium and
with 1% glucose (BHIg).
used for antibiofilm activity assays as described above with few
modifications. In a 96-well microtiter plate containing 20 mL
(~108 CFU/mL) of each reference strain culture was inoculated with
2.2. Metabolite extraction
the BHIg containing the diluted extracts or in BHIg without addition
of the extracts (negative control wells). All tests were carried out in
Metabolite extraction was performed as previously described
triplicate and the plate was incubated aerobically for 24 h at 37  C.
[15] with minor modifications. Briefly, total biomass obtained
After incubation plates were treated as described in section 2.3.1.
from a 500 mL culture in BHI medium was centrifuged (11,000 
The extracts diluted in BHIg were also evaluated for their ca-
g for 15 min). The supernatant was collected and thoroughly
pacity to dissociate mature biofilm of the reference strains, as
mixed with 250 mL of ethyl acetate (Tedia, OH, USA) in a sepa-
described for the cell-free supernatant in the previous section.
ration funnel and partitioned overnight into ethyl acetate and
The minimum biofilm eradication concentration (MBEC) was
aqueous phases. After removal of the upper organic phase,
determined considering the lowest concentration of each extract
partition of the aqueous phase was repeated twice with 250 mL
capable of inhibiting biofilm formation or dissociating mature
each of ethyl acetate. The pooled organic phases were evaporated
biofilm calculated by applying the formula shown in section 2.3.1.
to dryness in a rotary vacuum evaporator (HeiVap, Heidolph,
Schwabach, Germany) and the weight was determined. The
delipidated aqueous phase was filtered (Millipore 0.22 mm), 2.3.3. Statistical analysis
lyophilized and weighed. For the assays, the lipid extract was For biofilm production, statistical analyses were performed on
dissolved in BHIg supplemented with 10% dimethyl sulfoxide raw optical density data of three biological replicates. An unpaired
(DMSO) at a concentration of 5 mg/mL and the lyophilized Student’s t test was performed to determine the p-values between
aqueous residue at the same concentration in BHIg medium. control and treatment by GraphPad Prism 8 (GraphPad Software,
2
S.O. Nunes, H.S. Rosa, A.L.B. Canellas et al. Research in Microbiology xxx (xxxx) xxx

Inc., CA, USA). Results with a p-value  0.05 were considered sta- In addition to inhibiting biofilm formation, the Enterobacter 84.3
tistically significant. culture supernatant showed a high capacity to dissociate mature
biofilm formed by Staphylococcus spp. strains. The biofilm eradi-
2.4. Confocal microscopic observation of antibiofilm activity cation levels were similar to those observed for inhibitory activity
of biofilm formation. Eradication activity was 86.1% for S. aureus
In order to directly observe the multicellular structures in the ATCC 25923, 85.3% for S. aureus ATCC 29213 and 76.1% for
biofilm in the presence or absence of bioactive extract, Confocal S. epidermidis ATCC 35984 (Fig. 2).
Laser Scanning Microscopy (CLSM), with specific fluorescent
markers using the Filmtracer™ LIVE/DEAD™ Biofilm Viability Kit 3.2. Antibiofilm activity of aqueous extract
(Thermo Fisher Scientific, MA, USA) was performed according to
the instructions of the manufacturer. As previously described, ali- To investigate whether the antibiofilm activity of cell-free su-
quots of overnight culture (~108 CFU/mL) of S. aureus ATCC 25923 pernatant from Enterobacter 84.3 culture might be due to the lipidic
strain were distributed in chamber slides (Nunc Inc., IL, USA) for or aqueous fraction, the respective fractions were evaluated at
CLSM observations. After incubation at 37  C for 24 h, bacteria that different concentrations. No inhibition or dissociation effect on the
remained in suspension were removed by aspiration and the biofilms was observed for the lipid extract. However, the aqueous
remaining adherent cells on the slide were washed with sterile extract acted by inhibiting and dissociating biofilms of the refer-
distilled water and treated with the bioactive extract at the previ- ence strains at all concentrations tested (from 1 to 1024 mg/mL) and
ously established MBEC. Untreated biofilm control was included on antibiofilm activity rates and the MBEC are listed in Table 1.
each slide. After heat fixation, cells were incubated with SYTO 9 and For all reference strains, the aqueous extract inhibited biofilm
propidium iodide nucleic acid stains provided in the Filmtracer™ formation at a high percentage (Fig. 3AeC). S. aureus ATCC 25923
LIVE/DEAD™ Biofilm Viability Kit. Samples were observed on a biofilm formation was inhibited by 94.0% in the presence of the
Zeiss Axioplan microscope (Carl Zeiss, Jena, Germany) equipped for 16 mg/mL of the aqueous extract and it was dissociated by 85.3%
fluorescence microscopy. Live bacteria with intact cell membranes when treated with 32 mg/mL of the same bioactive extract.
show green fluorescence and dead bacteria with compromised Furthermore, a strong inhibitory effect (77%) on biofilms has also
membranes show red fluorescence. been observed against S. aureus ATCC 29213 and S. epidermidis ATCC
35984, as well as promising disruption rates (68%) of the mature
2.5. Cytotoxicity bioassays biofilms.
The aqueous extract showed a concentration-dependent anti-
A microassay for cytotoxicity in the L929 cell line (mouse biofilm activity both for the inhibitory effect on biofilm formation
fibroblast) was performed with the method known as “dye-up- and for the dissociation effect of mature biofilm from S. aureus and
take”, using neutral red dye [17], with minor modifications. One- S. epidermidis. Fig. 3D represents this observation on the mature
hundred microliters of L929 cell line suspension at a concentra- biofilm of S. aureus ATCC 25923. The effect of aqueous extract at
tion of 5  105 cells/mL were seeded in 96-well microplates. Cells 32 mg/mL on pre-formed S. aureus ATCC 25923 biofilm was also
were incubated for 24 h at 37  C in a 5% CO2 atmosphere to allow visualized on glass surfaces by confocal laser scanning microscopic
attachment. The active extract was added to the cell culture at imaging. This approach confirmed that the aqueous extract signif-
concentrations ranging from 500 mg/mL to 7.8 mg/mL, and the cells icantly reduced the biofilm biomass, average thickness, and sub-
were incubated for 24 h at 37  C in a 5% CO2 atmosphere. Then, strate coverage as compared to the untreated control (Fig. 4), thus
100 mL of 0.01% neutral red solution were added for 2 h at 37  C in a having a major effect on mature biofilm.
5% CO2 atmosphere. After incubation, the medium was removed
and the cell monolayers washed with PBS and the dye incorporated 3.3. Cytotoxicity of the active extract
by the viable cells was eluted using a mixture of methanol/acetic
acid/water (50:1:49). The dye uptake was determined by The aqueous extract was subjected to cytotoxicity assay in order
measuring the optical density of the eluate at 490 nm in an auto- against the mammalian L929 cell line. When the cells were treated
matic spectrophotometer (ELx800TM, Bio-TeK Instruments, Inc., with concentration from 7.8 to 500 mg/mL, the observed CC50 was
VT, USA). The 50% cytotoxic concentration (CC50) was defined as the higher than the maximum concentration tested (where CC50 stands
concentration of the extract which caused 50% reduction in the for 50% cytotoxic concentration, defined as the concentration
number of viable cells. required to reduce the cell number by 50% compared with that for
the untreated controls). These results suggest that the antibiofilm
3. Results substance present in the aqueous extract from Enterobacter 84.3 is
not toxic for L929 cells. Thus, as the CC50 for mammalian cells was
3.1. Effects of enterobacter 84.3 supernatants on biofilms higher than the observed MBEC (16e128 mg/mL) for S. aureus
strains, the use of this substance for inhibiting or dissociate biofilms
Antibiofilm activity was evaluated by microtiter plate assay could be recommended in the future.
using the cell-free supernatants obtained from Enterobacter strain
84.3 cultures. The results indicated that the marine strain produces 4. Discussion
substances able to reduce biofilm formation and to dissociate
mature biofilm formed by the tested indicator strains. For all Staphylococci are currently the most common cause of noso-
reference strains, the cell-free supernatant inhibited biofilm for- comial infections, mainly related to orthopaedic devices on which
mation at a high percentage, with a maximum value of 93.4% for the bacteria form biofilm [3e5]. Staphylococcal biofilms are pre-
S. aureus ATCC 25923, followed by 93.0% for S. aureus ATCC 29213 served from host defenses and often display dramatic decrease in
and of 89.0% for S. epidermidis ATCC 35984 (Fig. 1AeC). Biofilm antimicrobial susceptibility, resulting frequently in the develop-
formation by the three clinical strains isolated from orthopaedic ment of persistent and chronic infections [3,5].
prosthetic or surgical site infections was also inhibited and ranged In the past years many antibiofilm agents have been identified
from 80.1% for S. aureus 11123a over 82.9% for S. aureus 1117a to from various sources, including from sponge-associated bacteria,
66.2% for S. epidermidis 24 (Fig. 1DeF). which represent promising producers of substances for
3
S.O. Nunes, H.S. Rosa, A.L.B. Canellas et al. Research in Microbiology xxx (xxxx) xxx

Fig. 1. Antibiofilm activity of the cell-free supernatant from Enterobacter sp. 84.3 culture on Staphylococcus spp. biofilm formation. Biofilm produced in absence of supernatant was
used as control. The inhibition of biofilm formation is expressed as optical density of each well measured at 570 nm (A and D), *p < 0.0001. The ratio of biofilm absorbance/
planktonic absorbance was calculated and this value used to calculate the “biofilm formation” in percentage on the y axis (C and F). Wells of 96-well plate showing biofilms in the
absence and presence of supernatant after crystal violet staining (B and E). (For interpretation of the references to color in this figure legend, the reader is referred to the Web
version of this article.)

biotechnological and pharmaceutical applications [7e10,18e21]. inhibiting biofilm development and disaggregating the mature
However, none of them has entered the market. Hence, there is a biofilm, but without killing the Staphylococcus strains or inhib-
large unmet need for the development of antibiofilm formulations iting their growth. S. aureus and S. epidermidis infections associ-
to tackle this problem [7]. ated with biofilm development are estimated to reach 250,000
In a previous study, antibacterial tests were performed with cases per year in the USA with a mortality rate of up to 25%.
bacteria isolated from Oscarella sponges and their cell-free cul- These infections represent an important social and economic
ture supernatants [11], and the marine strain Enterobacter 84.3 burden worldwide [2]. The interest in the development of
was selected because no bacteriostatic or bactericidal activity innovative approaches for prevention and treatment of staphy-
against the target strains were observed, in spite of strong anti- lococcal adhesion and biofilm formation capabilities has there-
biofilm activity. The current study aimed to analyze for the first fore increased. A viable approach should target the
time the antibiofilm activities of sponge-associated Enterobacter staphylococcal adhesive properties without affecting bacterial
84.3 on staphylococcal biofilms. Our findings showed that viability in order to avoid the rapid appearance of escape mu-
Enterobacter 84.3 substances were particularly effective at both tants [21,22].

4
S.O. Nunes, H.S. Rosa, A.L.B. Canellas et al. Research in Microbiology xxx (xxxx) xxx

Fig. 2. Antibiofilm activity of the cell-free supernatant from Enterobacter sp. 84.3 culture on mature biofilms. Percentages of biofilm biomass reduction of reference staphylococcal
strains (A). Wells of 96-well plate showing mature biofilms untreated and treated after crystal violet staining (B). Control wells: 1. sterile growth medium (BHIg); 2. S. aureus ATCC
25923, indicator strain producing strong biofilm; 3. only cell-free supernatant. Test wells: 4. S. aureus ATCC 25923; 5. S. aureus ATCC 25923 treated with cell-free supernatant; 6.
S. aureus ATCC 29213; 7. S. aureus ATCC 29213 treated with cell-free supernatant; 8. S. epidermidis ATCC 35984; 9. S. epidermidis ATCC 35984 treated with cell-free supernatant.
Values below the wells 5, 7 and 9 show the percentages of mature biofilm biomass reduction. (For interpretation of the references to color in this figure legend, the reader is referred
to the Web version of this article.)

Table 1 Confocal laser scanning microscopy images also confirmed that

Antibiofilm activity of the aqueous extract from Enterobacter sp. 84.3 culture on the bioactive extract was able to dissociate the mature biofilm. This
staphylococcal biofilm.
characteristic shows that the antibiofilm agent could be applied
Strains Antibiofilm activity both for prevention of formation and removal of mature biofilm on
Inhibitory ratea (MBECb - mg/mL) indwelling medical devices. In addition, its use could also be
employed in a combination therapy with antibiotics of interest,
Inhibition Dissociation
since the bacterial cells will end up unprotected from the extra-
S. aureus ATCC 25923 94.0% (16) 85.3% (32) cellular matrix (biofilm) [21,22]. This model of action was also
S. aureus ATCC 29213 87.3% (64) 79.6% (128)
S. epidermidis ATCC 35984 77.0% (128) 68.2% (256)
proposed for pentadecanoic acid, a molecule derived from penta-
a
decanal produced by the Antarctic bacterium Pseudoalteromonas
Biofilm reduction rate (%).
b haloplanktis TAC125 [21,24]. Pentadecanoic acid is a good candidate
Minimum Biofilm Eradication Concentration (MBEC).
for combined therapies with conventional antibiotics, because it
prevented staphylococcal biofilm formation and positively modu-
Our results showed high rates of antibiofilm activity (from 66% lated the antimicrobial activity of vancomycin [21].
to 94%), including clinical strains isolated from orthopaedic In the case of infections related to orthopaedic prosthetic bio-
prosthetic and surgical site infections [13,14]. Sayem and col- film, the cost is very high since it involves long hospitalization time,
leagues [10] reported that cell-free supernatants obtained from surgery to change the prosthesis and antimicrobial administration
sponge associated Bacillus licheniformis SP1 showed inhibition as prophylaxis agents of nosocomial infections [3]. Biofilm dispersal
effects of almost 90% on biofilm formation by S. aureus. Interest- agents generally interfere with chemical pathways or processes,
ingly, S. aureus biofilms were more affected than S. epidermidis such as quorum sensing, which are required for bacteria to maintain
biofilms, as also observed in our study. In general, the cell-free the mode of existence as biofilm [5,25]. As dispersed cells are
supernatant from Enterobacter 84.3 culture inhibited S. aureus generally more susceptible to antimicrobial treatment than
biofilm formation by more than 75%, while acting on S. epidermidis biofilm-residing cells, this strategy has recently become an intense
biofilm at a 10% lower rate. This result could be explained by area of study [1,12,21].
differences in the extracellular matrix components of each strain, Based on the findings, we hypothesize that antibiofilm sub-
which comprise a complex mixture of polysaccharides, lipids, stances present in aqueous extract might interfere with the cell
extracellular DNA (eDNA) and proteins [1,20]. Studies have surface, thus influencing cellecell interactions as pre-requisite for
revealed how distinct constituents of the biofilm matrix biofilm formation [23], or with other steps of biofilm assembly.
contribute to its architectural stability and functionality. These Since the antibiofilm substances are found in the aqueous phase,
findings will provide the basis for developing novel therapeutics they might be polysaccharides. Polysaccharides have been previ-
that can effectively target components of the biofilm matrix and ously reported to produce anti-adherence effects between micro-
modulate biofilm stability [1,5,23]. organisms and surfaces [10,23].
In this study, we observed that the biofilm degradation activity In conclusion, the cell-free culture supernatant from sponge-
from Enterobacter 84.3 was caused by water-soluble substances. associated Enterobacter 84.3 contains water-soluble molecules
The aqueous extract presented minimum biofilm eradication con- that can provide a tool for better exploration of novel antibiofilm
centration values smaller than the CC50 observed for L929 cells substances. Inhibiting biofilm formation and dissociating mature
(higher than 500 mg/mL). These are some of the features that make biofilm of strains of S. aureus and S. epidermidis without affecting
it of great biotechnological interest. their growth represent a special feature of the antibiofilm activity

5
S.O. Nunes, H.S. Rosa, A.L.B. Canellas et al. Research in Microbiology xxx (xxxx) xxx

Fig. 3. Antibiofilm activity of the aqueous extract from Enterobacter sp. 84.3 culture on Staphylococcus spp. biofilm formation. The assays were performed at minimum biofilm
eradication concentration (MBEC) for each reference strain and the activity is concentration-dependent. The inhibition of biofilm formation is expressed as optical density of each
well measured at 570 nm (A), *p < 0.0001. Wells of 96-well plate showing biofilms in the absence and presence of aqueous extract after crystal violet staining (B). The absorbance
values were used to calculate the antibiofilm activity in percentage applying the formula shown in section 2.3.1(C). MBEC was defined at 32 mg/mL on pre-formed S. aureus ATCC
25923 biofilm (D). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

6
S.O. Nunes, H.S. Rosa, A.L.B. Canellas et al. Research in Microbiology xxx (xxxx) xxx

Fig. 4. Representative confocal laser scanning microscopy images of S. aureus ATCC 25923 biofilm in the presence of the bioactive aqueous extract (32 mg/mL), visualized by
fluorescence vital dye. 3D read out of the stained biofilm: (top) untreated biofilm control, and (bottom) treated biofilm. Biofilm incubated with SYTO 9 and propidium iodide nucleic
acid stains (A and C) and with FilmTracer stain (B and D). Green cells and clusters indicate bacterial cells with intact membranes (live). (For interpretation of the references to color
in this figure legend, the reader is referred to the Web version of this article.)

described in this report. Further research on such bioactive aqueous [10] Sayem S, Manzo E, Ciavatta L, Tramice A, Cordone A, Zanfardino A, et al. Anti-
biofilm activity of an exopolysaccharide from a sponge-associated strain of
extract might help developing new antibiofilm agents active
Bacillus licheniformis. Microb Cell Factories 2011;10:74.
against staphylococcal biofilms on indwelling medical devices. [11] Laport MS, Bauwens M, de Oliveira Nunes S, Willenz P, George I, Muricy G.
Culturable bacterial communities associated to Brazilian Oscarella species
(Porifera: Homoscleromorpha) and their antagonistic interactions. Antonie
Declaration of competing interest
Leeuwenhoek 2017;110:489e99.
[12] Verderosa AD, Totsika M, Fairfull-Smith KE. Bacterial biofilm eradication
No conflict of interest is declared. agents: a current review. Front Chem 2019;7:824.
[13] Schuenck RP, Cavalcante FS, Emery E, Giambiagi-de Marval M, dos Santos KR.
Staphylococcus aureus isolates belonging to different multilocus sequence
Acknowledgements types present specific virulence gene profiles. FEMS Immunol Med Microbiol
2012;65:501e4.
This work was supported by the Fundaça ~o Carlos Chagas Filho [14] Iorio NL, Caboclo RF, Azevedo MB, Barcellos AG, Neves FP, Domingues RM,
et al. Characteristics related to antimicrobial resistance and biofilm formation
de Amparo  a Pesquisa do Estado do Rio de Janeiro (E-26/ of widespread methicillin-resistant Staphylococcus epidermidis ST2 and ST23
010.101056/2018); Coordenaça ~o de Aperfeiçoamento de Pessoal de lineages in Rio de Janeiro hospitals, Brazil. Diagn Microbiol Infect Dis 2012;72:
Nível Superior - Brasil (Finance Code 001). 32e40.
[15] Santos OCS, Soares AR, Machado FL, Romanos MT, Muricy G, Giambiagi-
deMarval M, et al. Investigation of biotechnological potential of sponge-
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