Curr Microbiol (2016) 72:518–528
DOI 10.1007/s00284-015-0981-9
In Vitro Antibiofilm Activity of an Exopolysaccharide
from the Marine Thermophilic Bacillus licheniformis T14
Antonio Spanò1 • Pasqualina Laganà2 • Giuseppa Visalli2 • Teresa L. Maugeri1
Concetta Gugliandolo1
Received: 25 October 2015 / Accepted: 28 November 2015 / Published online: 11 January 2016
 Springer Science+Business Media New York 2016
Abstract The development of antibiofilm strategies is of
major interest in contrasting bacterial pathogenic biofilms. A
novel fructose and fucose rich exopolysaccharide (EPS1-T14)
produced by the recently described thermophilic Bacillus
licheniformis T14, isolated from a shallow hydrothermal vent
of Panarea Island (Eolian Island, Italy), was evaluated for its
effects on biofilm formation by multiresistant clinical strains
of Escherichia coli, Klebsiella pneumoniae, Pseudomonas
aeruginosa, and Staphylococcus aureus. The antibiofilm
activity of EPS1-T14 was assessed by microtiter plate assays
and visualized by confocal laser scanning microscopic ima-
ges. EPS1-T14, with molecular weight of 1000 kDa, reduced
biofilm formation on abiotic surfaces without affecting bac-
terial vitality. The novel EPS1-T14 is a water-soluble, non-
cytotoxic exopolymer able to prevent biofilm formation and
its use may represent a promising therapeutic strategy for
combating bacterial biofilm-associated infections. EPS1-T14
as antiadhesive biomolecule could be useful for novel
prospective in medical and nonmedical applications.
& Concetta Gugliandolo
cgugliandolo@unime.it
1
Research Centre for Extreme Environments and
Extremophiles, Department of Biological and Environmental
Sciences, University of Messina, V.le F. Stagno d’Alcontres
31, 98166 Messina, Italy
2 which bacterial biofilms have found to be involved include
Department of Biomedical Sciences and Morphological and
Functional Images, University of Messina, V. Consolare
Valeria, 98125 Messina, Italy
123
•
Introduction
Bacterial biofilms are cellular aggregates or surface-at-
tached microorganisms built in a matrix composed by
hydrated extracellular polymeric substances [11]. Bacterial
polysaccharides are the most abundant components, gen-
erally representing from 40 to 95 % of the extracellular
polymers [17]. Bacterial exopolysaccharides (EPSs) are the
major constituents of biofilms; they allow fix cells to solid
surfaces and to maintain the proper hydration and
micronutrients availability.
Several special characteristics of bacterial EPSs, such as
water solubility, biodegradability, biocompatibility,
bioadhesivity, mechanical and chemical resistance, swel-
ling and gelling power make them useful in different
industrial applications as emulsifiers, stabilizers, binders,
gelling agents, lubricants and thickening agents [5, 30, 33,
40].
Although bacterial aggregations have considerable
advantages in terms of self protection for the microbial
community, increasing microbial tolerance to exogenous
stress and the ability to survive in the presence of antibi-
otics or other biocides, biofilm formation has been shown
to cause many serious problems in different fields, such as
food spoilage, biofouling, and ineffective treatment for
chronic and recurrent infections [31].
Biofilm formation by bacterial pathogens represents a
serious concern in public health and medicine, since
65–80 % of all microbial diseases are biofilm based [15,
16]. Bacteria embedded in biofilms can be up to 1000-fold
more resistant to antibiotic treatment than the same
organism growing as free living [23]. Infection diseases for
lung infections of cystic fibrosis, urethritis, otitis, peri-
odontitis, and endocarditis [32, 36].
A. Spanò et al.: In Vitro Antibiofilm Activity of an Exopolysaccharide from the Marine…
Biofilm formation is a dynamic process consisting of
five steps: (1) initial attachment, (2) irreversible attach-
ment, (3) early development of biofilm architecture, (4)
maturation, and (5) dispersion [37, 49]. The adhesion to
surface becomes irreversible when the cells begin to
secrete EPSs [18]. The quorum sensing mechanism is
involved in biofilm formation and also in maintaining the
structure intact [14].
The knowledge of these steps can help understand how
to contrast the biofilm formation and its development.
Therefore, searching for compounds or strategies to pre-
vent the initial bacterial adhesion and the biofilm growth is
essential for human health, as well as for industrial and
food-processing activities.
Marine microorganisms, as free living or associated with
different organisms, represent until now untapped sources of
valuable biomolecules useful in different applications. Cul-
ture supernatants obtained from several marine bacteria
showed antibiofilm effects [49], and their active compounds
range from furanones, leading candidates reported as quo-
rum sensing inhibitors, to complex polysaccharides [52].
The metabolites produced by marine actinomycetes were
reported to have antibiofilm activity against Vibrio in marine
ecosystem [58] and a drug-resistant Staphylococcus aureus
[3]. Extracts from the coral associated actinomycetes [42]
and the Bacillus horikoshii [55] inhibit biofilm formation by
Streptococcus pyogenes. The marine bacilli, including the
Bacillus pumilus S6-15, were reported to inhibit the biofilm
formation in Gram-positive and Gram-negative species [41].
The supernatant designated SP1, produced by Bacillus
licheniformis associated to a marine sponge, was able to
reduce the initial adhesion and the biofilm development of
Escherichia coli PHL628 and Pseudomonas fluorescens,
other than of a range of Gram-positive and Gram-negative
bacteria [51]. The exoproducts of marine Pseudoal-
teromonas impair biofilm formation by a wide range of
pathogenic strains [13, 29]. The antibiofilm activity of the
supernatant of polysaccharide nature, derived from the
Antarctic marine Pseudoalteromonas haloplanktis TAC125,
has been reported against different staphylococci [44].
Recent findings suggest that some EPSs produced by
marine bacteria also possess the ability to negatively con-
trol biofilm-associated infections, such as the exopolysac-
charide A101 from the marine Vibrio sp. QY101 [25].
Our previous investigations demonstrated that new bacilli
isolated from shallow submarine hydrothermal vents of
Panarea Island (Italy) are able to produce EPSs useful in
biotechnological applications [21]. We recently described a
novel haloalkaliphilic, thermophilic Bacillus licheniformis
strain T14 isolated from Panarea Island, easy to cultivate in
short time in a wide range of temperature, pH, and salinity,
able to produce a new EPS (EPS1-T14) in large quantity by
using a relatively simple purification process [54]. EPS1-
519
T14 possesses interesting chemical and rheological charac-
teristics, and it represents a promising biomolecule in
pharmaceutical applications, as natural antiviral, immunos-
timulatory and immunomodulatory agents [22].
In this study, the exopolymer EPS1-T14 was evaluated for
the ability to inhibit the biofilm formation by multiresistant
clinical strains of Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aeruginosa, and Staphylococcus aureus.
Materials and Methods
Clinical Strains: Isolation, Identification,
and Antibiotic Resistance
The clinical strains are part of the collection at the
microbiological laboratory, internal to Department of
Biomedical Sciences and Morphological and Functional
Images, Section of Medicine Preventive, University of
Messina (Italy). Strains were chosen on the basis of their
strong antibiotic resistance and ability to produce biofilm
under standard conditions.
Clinical strains and their origins were: Escherichia coli
463 was isolated from diabetic ulcer at the foot of a
71-year-old man; Klebsiella pneumoniae 2659 from the
inner lumen of a bronchoscope; Pseudomonas aeruginosa
445 from a necrotic bedsore in a 77-year-old woman; and
Staphylococcus aureus 210 isolated from surgical wound
of a 73-year-old man.
The strains were isolated from the different clinical
specimens onto cultural media commonly used in micro-
biological analyses, including Columbia Blood Agar, Cled
Agar, and Mannitol Salt Agar (Oxoid, Basingstoke
Hampshire, United Kingdom). The isolates were identified
by using test strips of API 20NE, API 20E, and API Staph
systems (bioMérieux, Marcy l’Etoile, France), according to
the manufacturer’s instructions.
After growing overnight at 37 C into Brain Hearth
Infusion (Oxoid), the strains were tested for resistance or
sensitivity to different antibiotics, using the standard disk
diffusion method (Kirby Bauer test) and commercially
available antibacterial disks (Oxoid). For the disk diffusion
assay, bacteria were grown at 37 C for 24 h onto plates of
Tryptic Soy Agar (Oxoid), harvested and then suspended in
sterile water adjusted to a 0.5 McFarland turbidity standard
(bioMérieux), corresponding to 1.5 9 108 CFU ml-1. Sus-
pensions were inoculated in triplicate onto plates of Mueller-
Hinton agar using a cotton swab. After 24 h of incubation at
37 C, the diameter of inhibition zones was measured with a
precision caliper (Mitutoyo, Andover, UK).
Some antibiotics have been indifferently tested for both
Gram-negative and Gram-positive strains, despite their
exclusive use towards one or the other type of bacteria.
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520 A. Spanò et al.: In Vitro Antibiofilm Activity of an Exopolysaccharide from the Marine…
Each bacterial strain was classified as resistant (R), inter-
mediate resistant (I), or susceptible (S) according to the
breakpoints established by Clinical Laboratory Standards
Institute (CLSI, 2006) [9].
Bacillus licheniformis Strain T14 Producer
of EPS1-T14
Bacillus licheniformis strain T14 as producer of the novel
EPS1-T14 has been previously described [54]. Briefly, the
strain was isolated from a thermal fluid sample collected at
8 m depth from Bottaro vent, located off the eastern coast of
Panarea Island (Eolian Islands, Italy). Characteristics of the
emitted fluid were temperature 50 C, pH 5.42, and conduc-
tivity 42.90 mS m-1. The strain grew aerobically from 25 to
60 C and its optimal temperature occurred at 50 C. The pH
range for growth was 4–10, with optimum at pH 8. Strain T14
grew in a range 2–10 % NaCl with optimal growth at 5 %. The
exopolysaccharide was obtained at the beginning of the sta-
tionary growth phase of the strain in the minimal medium
SWY (yeast extract, 0.1 %, sucrose 5 %, in seawater) after
48 h of incubation in bioreactor under optimal growth con-
dition (temperature 50 C, pH 8, and NaCl 5 %). The purified
fraction EPS1-T14 contained fructose/fucose/glucose in a
relative proportion of 1.0:0.75:0.28 and galactosamine and
mannose as contaminants. It possessed a molecular weight of
1000 kDa, displaying a trisaccharide unit constituted by
sugars with a b-manno-pyranosidic configuration.
Measurement of the Reduction of Surface Tension
and the Emulsifying Activity of EPS1-T14
The reduction of the surface tension and the emulsifier
activity (E24) of the EPS1-T14 were tested. The surface
tension of the EPS1-T14 solution (0.5 %, w/v) was deter-
mined with a digital tensiometer K10T (Krüss, Hamburg,
Germany) by using Wilhelmy Plate method, which measures
the weight of the liquid drawn by a plate when a plate is lifted
from or through the surface of liquid. The weight of the liquid
is proportional to the surface tension of the liquid [26].
To test the emulsifying activity, 2 ml of an EPS1-T14
aqueous solution (0.5 %, w/v) was mixed with an equal
volume of kerosene in a glass tube (10 cm high and 1 cm
in diameter) and stirred in the vortex at high speed for
2 min. The emulsion and aqueous layers were measured
after 24 h, and emulsification index (E24) was calculated by
the following formula: E24 = (Volume of the emulsion
layer/Total volume) 9 100.
Biofilm Production
To evaluate the biofilm formation by the clinical strains,
the assay was carried out in 96-well polystyrene microtiter
123
plates [10]. Overnight cultures in peptone water of each
strain were diluted up to the OD600 = 0.1 (equivalent to
1.5 9 108 bacteria ml-1), and wells of microtiter plates
were filled (eight replicates) with aliquots of 100 ll each.
The plates were incubated at 37 C for 24 h without
shaking for biofilm production, and nonadherent bacteria
were removed by washing 5 times with distilled water, by
using a Plate Washer (Das Instrument, Italy). The adherent
bacteria (biofilms) were stained with 1 % crystal violet
solution (w/v) for 45 min [43]. Excess stain was removed
by aspiration, and the plates were washed (5 times) and air
dried (for 45 min). The stained biofilms were solubilized
with ethanol 99 %. Biofilm mass, estimated by the level of
the crystal violet present in the destaining solution, was
spectrophotometrically (OD = 600 nm) determined using
a microtiter plate reader (Multiskan GO, Thermo Scientific,
USA). The average OD from the control wells was sub-
tracted from the OD of all tested wells.
Antibiofilm Assays
Pre-screening: Antibiofilm Effects of Strain T14
Supernatants
In a preliminary step, supernatants of B. licheniformis
strain T14 from two cultural media, differing in nutritional
sources, were evaluated for their capacity to inhibit biofilm
formation of the clinical strains. Strain T14 was inoculated
into Bacto Marine Broth 2216 (Difco Laboratories, Detroit,
MI) (MB) and into SWY containing yeast extract (0.1 %,
w/v) plus sucrose 5 % as carbon source. The cultures were
incubated under strain T14 optimal growth conditions
(temperature of 50 C, pH 8, and NaCl 5 %) for 48 h and
then centrifuged at 10,000g for 30 min to separate the cell
pellets from media. To remove all bacterial cells, super-
natants were filtered through a 0.2-lm-pore-size membrane
(Sartorius), and to ensure that no cells were present in the
filtrates, 100 ll was spread onto plates of Bacto Marine
Agar 2216 (Difco Laboratories, Detroit, MI).
In order to enhance the antibiofilm activity, different
concentrations of cell-free supernatants (CFS) were used,
including also the addition of tenfold concentrated super-
natants (CFSC).
To obtain CFSC, an aliquot (10 ml) of each supernatant
was lyophilized and dissolved in 1/10 of sterile distilled
water (resulting in 10 9 concentration). In a 96-well
microtiter plate containing 100 ll of each strain culture
(OD600 = 0.1, equivalent to 1.5 9 108 bacteria ml-1), CFS
and CFSC were added at a final concentration of 1, 2, 4, 8
and 16 % (v/v). The reduction of biofilm formation of each
clinical strain was expressed as antibiofilm activity (%), by
applying the following formula: (ODcontrol - ODsample/
ODcontrol) 9 100. Each data point was averaged from eight
A. Spanò et al.: In Vitro Antibiofilm Activity of an Exopolysaccharide from the Marine…
replicated wells, and the standard deviation (SD) was
calculated.
Screening: Antibiofilm Activity of EPS1-T14
To evaluate the ability to inhibit bacterial biofilm formation
by clinical strains, overnight cultures (100 ll) of each strain
were added to a 96-well microtiter plate together with EPS1-
T14 at different final concentrations (25, 50, 100, 200, and
400 lg ml-1). The biofilm formation was assessed spec-
trophotometrically in the presence or without EPS1-T14.
The reduction of biofilm formation of each clinical strain
was expressed as antibiofilm activity (%), calculated as
follows: (ODcontrol - ODsample/ODcontrol) 9 100. Each data
point was averaged from eight replicated wells and the SD
was calculated.
Confocal Microscopic Observation of Biofilm
To directly observe the multicellular structures into the bio-
films, with or without the addition of EPS1-T14, the Confocal
Laser Scanning Microscopy (CLSM), with specific fluores-
cent markers, was used [6]. As previously described, aliquots
of overnight cultures in peptone water (adjusted to
OD600 = 0.1) were distributed in chamber slides (Nunc Inc.,
Naperville, Illinois) for CLSM observations. After incubation
at 37 C for 24 h, the bacteria that remained in suspension
were eliminated and the remaining adherent cells on the slide,
after washing with Phosphate-Buffered Saline (PBS), were
fixed by heat and finally stained with 20 lg ml-1 of propid-
ium iodide (PI) (Sigma), a nucleic acids’ intercalating and
fluorescent agent.
The slides were incubated in the dark at 30 C for 5 min
to enable the fluorescent labelling of the bacteria. The flu-
orescence excitation maximum is 535 nm and the emission
maximum is 617 nm. Observations were performed by
CLSM using a TCS SP2 microscope (Leica Microsystems
Heidelberg, Mannheim, Germany), equipped with Ar/Kr
laser, and coupled to a microscope (Leica DMIRB).
Antibacterial Activity
To investigate whether the inhibitory effects of supernatants
(CFS and CFSC) from strain T14 and of EPS1-T14 on
biofilm formation by clinical strains might be due to the
direct growth inhibition, antibacterial activity was evaluated.
Supernatants from strain T14 were tested by using the agar
diffusion method, as accepted by the National Committee for
Clinical Laboratory Standards (NCCLS 2000). Each clinical
strain was overnight grown into Tryptone Soya Broth (Difco,
Becton–Dickinson and Company, USA) for 24 h at 37 C.
Suspensions of each strain (equivalent to a 0.5 McFarland
standard) were inoculated in triplicate onto plates of
521
Tryptone Soya Agar (Difco). Aliquots of 20 ll of CFS and
CFSC were applied to sterile filter paper discs (bioMérieux,
6 mm in diameter). The disks were placed onto the inocu-
lated plates. Plates were incubated overnight at 37 C. The
diameter of complete inhibition zones was measured, and
means and standard deviations were calculated.
The antibacterial effects of EPS1-T14 on the clinical
strains cultures were observed by growth curve analysis.
Microtiter plates were filled (eight replicates) with over-
night cultures in peptone water (100 ll) of each clinical
strain (OD600 = 0.1), with the addition of EPS1-T14 at a
final concentration of 400 lg ml-1. The plates were incu-
bated at 37 C for 24 h without shaking. Bacterial cultures
without the addition of the EPS were used as control.
OD600 values were recorded for up to 24 h at 1-h intervals.
Results
Clinical Strains: Antibiotic Resistance
Antibiotic sensitivity/resistance pattern of the clinical
strains are reported in Table 1. Strains were resistant to the
major part of the tested antibiotics, and P. aeruginosa
strain was susceptible only to fosfomycin.
Effects of B. licheniformis T14 Supernatants
on Biofilms Formation
A preliminary assessment of the antibiofilm activity was
performed by the microtiter plate assay using the cell-free
supernatants obtained from strain T14 grown into different
culture conditions. The supernatant obtained by using
medium MB did not show any antibiofilm activity (data not
shown), while the cell-free supernatants derived from
medium SWY (plus 5 % of sucrose) (CFS and CFSC) were
effective against all the tested strains. These results indi-
cated that, differently from the composition of medium
MB, the high content of sucrose as a unique carbon source
in medium SWY greatly influenced the production from
strain T14 of biomolecules able to reduce the biofilm for-
mation from the tested strains.
The antibiofilm activity of the cell-free supernatants was
concentration dependent and differed among the tested
bacteria (Table 2). CFS at a concentration of 2 % showed a
strong antibiofilm activity (C50 % of biofilm reduction)
only against E. coli. At the highest concentration of CFS
(16 %), the percentage of biofilm reduction was strong for
all the target strains, with the only exception for K. pneu-
moniae (42 %), and it reached the maximum value (69 %)
for E. coli (Table 2).
An enhanced biofilm inhibition activity was observed by
adding the tenfold concentrated supernatants (CFSC)
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522 A. Spanò et al.: In Vitro Antibiofilm Activity of an Exopolysaccharide from the Marine…

Table 1 Antibiotic sensitivity pattern of the clinical strains used in this work
Escherichia Klebsiella Pseudomonas Staphylococcus
coli 463 pneumonia 2659 aeruginosa 445 aureus 210

Amikacin (30 lg) I I I

Ampicillin (10 lg) R R R R
Amoxicillin (10 lg) R
Amoxicillin ? clavulanic acid (20 lg ? 10 lg) R R R S
Azithromycin (15 lg) S I R S
Aztreonam (30 lg) R R R
Carbenicillin (100 lg) R R R R
Cephalexin (30 lg) I
Cefalotin (30 lg) S
Cefazolin (30 lg) R R R R
Cefotaxime (30 lg) R R R R
Cefoxitin (30 lg) I S R
Ceftazidime (30 lg) R R R
Ceftriaxone (30 lg) R R R R
Cefuroxime (30 lg) R R R S
Cinoxacin (100 lg) R R R R
Ciprofloxacin (5 lg) R R R S
Chloramphenicol (30 lg) S S R S
Clindamycin (2 lg) S
Colistin sulphate (10 lg) R R I
Doxycycline (30 lg) S
Erythromycin (15 lg) S
Fosfomycin (50 lg) R R S R
Gentamycin (10 lg) I R R R
Imipenem (10 lg) S S R
Josamycin (30 lg) S
Levofloxacin (5 lg) R R R S
Lincomycin (2 lg) R
Linezolid (10 lg) S
Mezlocillin (75 lg) R R R
Minocycline (30 lg) S
Nalidixic Acid (30 lg) R R R
Netilmicin (30 lg) I I R
Nitrofurantoin (300 lg) S S R S
Norfloxacin (10 lg) R R R S
Ofloxacin (5 lg) R R R S
Oxacillin (1 lg) R
Penicillin G (10 lg) R
Pipemidic Acid (20 lg) R R R
Piperacillin (100 lg) S R I R
Rifampicin (30 lg) R I I S
Sisomicin (30 lg) I R R
Sulphamethoxazole ? Trimethoprim S I R
(23.75 lg ? 1.25 lg)
Teicoplanin (30 lg) S
Tetracycline (30 lg) S I R I
Tigecycline (15 lg) I R R I
Tobramycin (10 lg) I S R
Vancomycin (30 lg) S

R resistant, I intermediate, S susceptible

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A. Spanò et al.: In Vitro Antibiofilm Activity of an Exopolysaccharide from the Marine… 523

Table 2 Pre-screening of antibiofilm activity: reduction in biofilm further increasing in concentration was particularly effec-
formation (%) of increasing concentrations of cell-free supernatants tive against E. coli, P. aeruginosa, and S. aureus (72, 61,
(CFS) from strain T14 on biofilm formation of clinical strains
and 66 % reduction, respectively).
Antibiofilm activity (mean %) When antibacterial test was performed with super-
A CFS concentration (v/v) natants, no inhibition halo surrounding the disks was
observed, indicating that the supernatants have no
1 ll 2 ll 4 ll 8 ll 16 ll
bacteriostatic or bactericidal activity against the target
Escherichia coli 463 11 50 60 64 69 strains.
Klebsiella pneumoniae 2659 7 24 28 42 47
Pseudomonas aeruginosa 445 4 4 12 49 52
Staphylococcus aureus 210 5 13 21 31 53 Antibiofilm Activity of EPS1-T14
B CSFc concentration (v/v)
The inhibition effects at increasing concentrations (from 25
1 ll 2 ll 4 ll 8 ll 16 ll to 400 lg ml-1) of EPS1-T14 on biofilm formation by the
Escherichia coli 463 68 67 67 72 72
tested clinical strains are reported in Fig. 1. The EPS1-T14
Klebsiella pneumoniae 2659 30 54 54 53 51
showed a dose-dependent inhibitory effect on biofilm for-
Pseudomonas aeruginosa 445 3 24 53 57 61
mation by E. coli and S. aureus, but not by K. pneumoniae
and P. aeruginosa, indicating that the antibiofilm effects
Staphylococcus aureus 210 45 60 60 66 65
depend on the dose applied and the strain tested.
(A) not concentrated CFS and (B) tenfold concentrated CFS (CFSC) In the presence of EPS1-T14 at 100 lg ml-1, a strong
reduction of the biofilm formation by E. coli (52 %) and K.
pneumoniae (55 %) was observed. By adding 200 lg ml-1
(Table 2). CFSC at 1 % showed a strong biofilm reduction of EPS1-T14, a further reduction in biofilm formation by
only for E. coli (68 %), whereas the addition at 2 % E. coli (73 %) was observed. The addition of 400 lg ml-1 of
resulted in a strong activity also against K. pneumoniae EPS1-T14 strongly reduced the biofilm formation by all the
(54 %) and S. aureus (60 %). CFSC used at 4 % showed a strains (E. coli 74 %, K. pneumoniae 56 %, P. aeruginosa
strong antibiofilm activity for all the target strains and a 54 %, and S. aureus 60 %). The antibiofilm activity of EPS1-

E. coli 463 K. pneumoniae 2659

100 100
Biofilm formation (%)

Biofilm formation (%)

80 80

60 60

40 40

20 20

0 0
C 25 50 100 200 400 C 25 50 100 200 400
EPS1-T14 (µg ml-1) EPS1-T14 (µg ml-1)

P. aeruginosa 445 S. aureus 210

100 100

80 80
Biofilm formation (%)
Biofilm formation (%)

60 60

40 40

20 20

0 0
C 25 50 100 200 400 C 25 50 100 200 400
EPS1-T14 (µg ml-1) EPS1-T14 (µg ml1)

Fig. 1 Screening of antibiofilm activity: inhibition effects of increas- The biofilm formation is expressed in percentage. Biofilm produced in
ing concentrations (from 25 to 400 lg ml-1) of EPS1-T14 on biofilm absence of EPS1-T14 was used as control (C)
formation by E. coli, K. pneumoniae, P. aeruginosa, and S. aureus.

123
524 A. Spanò et al.: In Vitro Antibiofilm Activity of an Exopolysaccharide from the Marine…
Control EPS1-T14 (400 µg ml-1)
E. coli 463
K. pneumoniae 2659
P. aeruginosa 445
S. aureus 210
Fig. 2 Confocal laser imagines (9400) of biofilm formed by E. coli
463, K. pneumoniae 2659, P. aeruginosa 445, and S. aureus 210 in
the absence (control) or presence of EPS1-T14 (400 lg ml-1)
T14 at 400 lg ml-1 was also visualized onto glass surfaces
by confocal laser scanning microscopic images (Fig. 2).
To evaluate whether the antibiofilm effects were related
to reduction of growth rate of the target strains, growth
curves were measured in the presence and absence of
EPS1-T14. The presence of EPS1-T14 at the highest con-
centration used (400 lg ml-1) did not influence the growth
rates of the tested clinical strains (Fig. 3), indicating that
the biopolymer have no antibacterial activity. These results
showed that bacterial biofilm inhibition did not due to a
direct reduction of growth rate of the target strains, but to
an inhibition of their biofilm formation.
Biosurfactant Activities of EPS1-T14
A significant reduction in surface tension (from 65.2 ± 3
to 42.4 ± 3 mN m-1) was recorded in the presence of
EPS1-T14 (0.5 %, w/v).
123
The emulsion forming and stabilizing capacity of EPS1-
T14 were tested on hydrocarbons in comparison with Tri-
tonX-100, which is a synthetic emulsifier. When compared
with an equal solution of TritonX-100 in the presence of
kerosene (E24 = 70.5 ± 1 %), the EPS1-T14 has proven to
possess a good emulsion capacity (E24 = 58.4 ± 2 %).
Moreover, emulsions were stable for several weeks sug-
gesting also a stabilizing effect.
Discussion
Novel bacteria associated with shallow hydrothermal vents
have demonstrated their ability to produce novel exopoly-
mers, which represent promising products for biotechno-
logical and pharmaceutical applications [1, 2, 21, 22, 34, 35,
38, 39]. Bacterial exopolysaccharides, other than biofilm
stabilization and energy storage, exhibit different properties
and functions greatly determined by their chemical com-
position, molecular structure, and average molecular weight.
The EPS1-T14, produced by the haloalkaliphilic, ther-
mophilic B. licheniformis T14, displayed a high carbohy-
drate content (99 %) and elevated molecular weight (about
1000 kDa); contained fructose and fucose as major
monosaccharides; and showed interesting chemical and
rheological characteristics. The EPS is soluble in water,
noncytotoxic up to 400 lg ml-1, stable at high tempera-
tures, with a degradation temperature of 240 C, the fea-
tures that make it of great biotechnological interest [54].
Until now, bacterial polysaccharides containing fucose
are very few and include clavan [56], fucogel [45], and
fucopol [19]. Their functional characteristics, such as
thickening, emulsion stabilizing, film forming, and floc-
culating capacities, have an increased interest in many
biotechnological applications [20, 50, 56]. Particular
attention was paid towards the medical and pharmaceutical
applications of fucose-containing polysaccharides, as
anticarcinogenic and antiinflammatory agents, useful in the
treatment of rheumatoid arthritis, in age-related patholo-
gies accompanied by tissue loss, in acceleration of wound
healing and as hydrating and antiaging additives [8, 46].
However, no antibiofilm activities of clavan, fucogel, and
fucopol have been reported until now.
Recently, we reported that EPS1-T14 is able to stimulate the
immune response and thus contribute to the antiviral immune
defense, acting as immunostimulant and immunomodulator
agent [22]. In this study, EPS1-T14 was found to reduce
in vitro biofilm formation of both Gram-negative and Gram-
positive, multiresistant clinical strains.
Sayem et al. [51] reported that cell-free supernatants
obtained from the sponge associated B. licheniformis SP1
showed inhibition effects on biofilm formation by E. coli
PHL628 and P. fluorescence (about 89 and 80 % of
A. Spanò et al.: In Vitro Antibiofilm Activity of an Exopolysaccharide from the Marine… 525

1.4
E. coli 463 K. pneumoniae 2659
1.4

1.2 1.2

1.0 1.0

0.8 0.8
OD600

OD600
0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h) Time (h)
Control EPS1-T14 Control EPS1-T14

P. aeruginosa 445 S. aureus 210

1.4 1.4

1.2 1.2

1.0 1.0

0.8 0.8

OD600
OD600

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h) Time (h)
Control EPS1-T14 Control EPS1-T14

Fig. 3 Growth curves of clinical strains in the absence (control) or presence of EPS1-T14 (400 lg ml-1)

inhibition rates, respectively), and the activity was sup-
posed to be related to the EPS produced in the cultural
medium.
Other few bacterial purified EPSs have been recently
found to possess antibiofilm activities against pathogenic
bacteria (Table 3). With the exception of the EPS1-T14
and PAM galactan [4], none of the listed EPSs possessed
antibiofilm effects against K. pneumoniae.
The EPSs Ec300p [48], Pel [47], and PI80 EPS [27]
were found active only against the biofilm formation by
Gram-positive bacteria. The EPS from Lactobacillus aci-
dophilus A4 [28] possessed inhibitory activity against both
Gram-positive and Gram-negative bacteria; however, its
concentration active against the biofilm formation from
E. coli and P. aeruginosa was much higher (1.0 mg ml-1)
than that reported for EPS1-T14. Although the EPS from
Lactobacillus plantarum YW32 [57] showed a wide range
of antibiofilm activities, its inhibition rates were lesser
(\50 %) than those observed for EPS1-T14.
A101, produced by the marine Vibrio sp. QY101 [25],
was more effective against P. aeruginosa (inhibition rate
75 %) and S. aureus (inhibition rate [ 90 %) than that
EPS1-T14 (54 and 60 %, respectively). On the other hand,
EPS1-T14 was more effective than A101 in contrasting the
biofilm formation by E. coli (inhibition rate 74 %), and it
was also more active in reducing the biofilm produced by
K. pneumoniae (inhibition rate 56 %).
The EPS1-T14 reduced the biofilm formation of all the
target pathogenic bacteria without antibacterial effects,
similarly to other EPSs previously reported [25, 52].
The mechanism of action of EPS1-T14 could be inde-
pendent from quorum sensing, since its chemical compo-
sition is unrelated to any of the so far known quorum
sensing molecules. However, it is possible that indirect
effects on cells may occur, which can alter the expression
of quorum sensing molecules, but these aspects need to be
further analyzed.
EPS1-T14 showed surfactant activities, modifying the
surface tension, emulsifying, and stabilizing hydrocarbon/
water emulsions. For its surfactant activities, it can be
supposed that EPS1-T14 could influence bacterial cell
surface hydrophobicity, and therefore, it could interfere
with the initial adhesion step, essential for successful bio-
film development. Among the biosurfactants, lipopeptides
(such as surfactin) represent a class of microbial surfactants
with remarkable surface properties and biological activi-
ties, such as surplus oil recovery, food-processing, de-
emulsification, antimicrobial, and antitumor, antiviral, and
antiadhesive activities [12, 53]. A lipopeptide biosurfactant
produced by Bacillus natto TK-1 has been reported to have
a strong surface activity, and it was found to be an anti-
adhesive and antimicrobial agent against several bacterial
strains [7]. The possible mechanism of this lipopeptide
biosurfactant against Salmonella typhimurium (maximum

123
526 A. Spanò et al.: In Vitro Antibiofilm Activity of an Exopolysaccharide from the Marine…

Table 3 Bacterial exopolysaccharides with antibiofilm activity against pathogenic bacteria

Species and strain Exopolysaccharide Molecular Main component Anti-biofilm activity Reference
weight (kDa) against strain

Bacillus licheniformis T14 EPS1-T14 1000 Fructose, Fucose E. coli This study
K. pneumoniae
P. aeruginosa
S. aureus
Escherichia coli Ec300 Ec300p [500 Mannose, Glucose, S. aureus [48]
Galactose, Glucuronic acid S epidermidis
Ent. faecalis
Kingella kingae PYKK081 PAM galactan [300 Galactose S. aureus [4]
K. pneumoniae
Kin. kingae
Lactobacillus acidophilus A4 r-EPS Unknown Unknown E. coli [28]
Sal. enteritidis
Sal. typhimurium
Y. enterocolitica
P. aeruginosa
Lis. monocytogenes
B. cereus
Lactobacillus plantarum YW32 EPS 103 Mannose, Fructose, Galactose, E. coli [57]
Glucose Shi. flexneri
S. aureus
Sal. typhimurium
Pseudomonas aeruginosa PAO1 Pel Unknown Glucose-rich S. epidermidis [47]
Streptococcus phocae PI80 PI80 EPS 280 Arabinose Lis. monocytogenes [27]
B. cereus
S. aureus
Vibrio sp. QY101 A101 546 Galacturonic acid, Glucuronic P. aeruginosa [25]
acid, Rhamnose, Glucosamine S. aureus
B Bacillus, E Escherichia, Ent Enterococcus, K Klebsiella, Kin Kingella, Lis Listeria, S Staphylococcus, Sal Salmonella, Shi Shigella, Y Yersinia

reduction in adherence 52 %) was the disruption of the
outer membrane cells.
The presence of fructose and fucose as major compo-
nents may provide additional biological properties to
EPS1-T14 compared to those composed of more common
sugar monomers. Fucose and fructose are inhibitors of the
surface lectins (proteins designed as fimbriae or pili) of
various bacteria, including P. aeruginosa [24]; therefore,
EPS1-T14 could interfere with the assembly of adhesins in
the cell wall, but this possibility needs further researches.
In conclusion, the EPS1-T14 possesses several inter-
esting features, including water-solubility, noncytotoxicity,
biodegradability, and ecological acceptability. Moreover,
its stability to elevated temperatures may confer it a great
efficiency in decreasing biofilm formation at different
temperature conditions. Therefore, EPS1-T14 could rep-
resent a more powerful and a better alternative to the
previously identified polysaccharides, suggesting novel
prospective in medical and nonmedical applications, as
antiadhesive drug.
Compliance with Ethical Standards
Conflict of interest The authors declare no conflict of interest.
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