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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 Introduction

major interest in contrasting bacterial pathogenic biofilms. A
novel fructose and fucose rich exopolysaccharide (EPS1-T14) Bacterial biofilms are cellular aggregates or surface-at-
produced by the recently described thermophilic Bacillus tached microorganisms built in a matrix composed by
licheniformis T14, isolated from a shallow hydrothermal vent hydrated extracellular polymeric substances [11]. Bacterial
of Panarea Island (Eolian Island, Italy), was evaluated for its polysaccharides are the most abundant components, gen-
effects on biofilm formation by multiresistant clinical strains erally representing from 40 to 95 % of the extracellular
of Escherichia coli, Klebsiella pneumoniae, Pseudomonas polymers [17]. Bacterial exopolysaccharides (EPSs) are the
aeruginosa, and Staphylococcus aureus. The antibiofilm major constituents of biofilms; they allow fix cells to solid
activity of EPS1-T14 was assessed by microtiter plate assays surfaces and to maintain the proper hydration and
and visualized by confocal laser scanning microscopic ima- micronutrients availability.
ges. EPS1-T14, with molecular weight of 1000 kDa, reduced Several special characteristics of bacterial EPSs, such as
biofilm formation on abiotic surfaces without affecting bac- water solubility, biodegradability, biocompatibility,
terial vitality. The novel EPS1-T14 is a water-soluble, non- bioadhesivity, mechanical and chemical resistance, swel-
cytotoxic exopolymer able to prevent biofilm formation and ling and gelling power make them useful in different
its use may represent a promising therapeutic strategy for industrial applications as emulsifiers, stabilizers, binders,
combating bacterial biofilm-associated infections. EPS1-T14 gelling agents, lubricants and thickening agents [5, 30, 33,
as antiadhesive biomolecule could be useful for novel 40].
prospective in medical and nonmedical applications. 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].
& Concetta Gugliandolo
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,
Research Centre for Extreme Environments and 16]. Bacteria embedded in biofilms can be up to 1000-fold
Extremophiles, Department of Biological and Environmental
more resistant to antibiotic treatment than the same
Sciences, University of Messina, V.le F. Stagno d’Alcontres
31, 98166 Messina, Italy organism growing as free living [23]. Infection diseases for
2 which bacterial biofilms have found to be involved include
Department of Biomedical Sciences and Morphological and
Functional Images, University of Messina, V. Consolare lung infections of cystic fibrosis, urethritis, otitis, peri-
Valeria, 98125 Messina, Italy odontitis, and endocarditis [32, 36].

A. Spanò et al.: In Vitro Antibiofilm Activity of an Exopolysaccharide from the Marine… 519

Biofilm formation is a dynamic process consisting of T14 possesses interesting chemical and rheological charac-
five steps: (1) initial attachment, (2) irreversible attach- teristics, and it represents a promising biomolecule in
ment, (3) early development of biofilm architecture, (4) pharmaceutical applications, as natural antiviral, immunos-
maturation, and (5) dispersion [37, 49]. The adhesion to timulatory and immunomodulatory agents [22].
surface becomes irreversible when the cells begin to In this study, the exopolymer EPS1-T14 was evaluated for
secrete EPSs [18]. The quorum sensing mechanism is the ability to inhibit the biofilm formation by multiresistant
involved in biofilm formation and also in maintaining the clinical strains of Escherichia coli, Klebsiella pneumoniae,
structure intact [14]. Pseudomonas aeruginosa, and Staphylococcus aureus.
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- Materials and Methods
vent the initial bacterial adhesion and the biofilm growth is
essential for human health, as well as for industrial and Clinical Strains: Isolation, Identification,
food-processing activities. and Antibiotic Resistance
Marine microorganisms, as free living or associated with
different organisms, represent until now untapped sources of The clinical strains are part of the collection at the
valuable biomolecules useful in different applications. Cul- microbiological laboratory, internal to Department of
ture supernatants obtained from several marine bacteria Biomedical Sciences and Morphological and Functional
showed antibiofilm effects [49], and their active compounds Images, Section of Medicine Preventive, University of
range from furanones, leading candidates reported as quo- Messina (Italy). Strains were chosen on the basis of their
rum sensing inhibitors, to complex polysaccharides [52]. strong antibiotic resistance and ability to produce biofilm
The metabolites produced by marine actinomycetes were under standard conditions.
reported to have antibiofilm activity against Vibrio in marine Clinical strains and their origins were: Escherichia coli
ecosystem [58] and a drug-resistant Staphylococcus aureus 463 was isolated from diabetic ulcer at the foot of a
[3]. Extracts from the coral associated actinomycetes [42] 71-year-old man; Klebsiella pneumoniae 2659 from the
and the Bacillus horikoshii [55] inhibit biofilm formation by inner lumen of a bronchoscope; Pseudomonas aeruginosa
Streptococcus pyogenes. The marine bacilli, including the 445 from a necrotic bedsore in a 77-year-old woman; and
Bacillus pumilus S6-15, were reported to inhibit the biofilm Staphylococcus aureus 210 isolated from surgical wound
formation in Gram-positive and Gram-negative species [41]. of a 73-year-old man.
The supernatant designated SP1, produced by Bacillus The strains were isolated from the different clinical
licheniformis associated to a marine sponge, was able to specimens onto cultural media commonly used in micro-
reduce the initial adhesion and the biofilm development of biological analyses, including Columbia Blood Agar, Cled
Escherichia coli PHL628 and Pseudomonas fluorescens, Agar, and Mannitol Salt Agar (Oxoid, Basingstoke
other than of a range of Gram-positive and Gram-negative Hampshire, United Kingdom). The isolates were identified
bacteria [51]. The exoproducts of marine Pseudoal- by using test strips of API 20NE, API 20E, and API Staph
teromonas impair biofilm formation by a wide range of systems (bioMérieux, Marcy l’Etoile, France), according to
pathogenic strains [13, 29]. The antibiofilm activity of the the manufacturer’s instructions.
supernatant of polysaccharide nature, derived from the After growing overnight at 37 C into Brain Hearth
Antarctic marine Pseudoalteromonas haloplanktis TAC125, Infusion (Oxoid), the strains were tested for resistance or
has been reported against different staphylococci [44]. sensitivity to different antibiotics, using the standard disk
Recent findings suggest that some EPSs produced by diffusion method (Kirby Bauer test) and commercially
marine bacteria also possess the ability to negatively con- available antibacterial disks (Oxoid). For the disk diffusion
trol biofilm-associated infections, such as the exopolysac- assay, bacteria were grown at 37 C for 24 h onto plates of
charide A101 from the marine Vibrio sp. QY101 [25]. Tryptic Soy Agar (Oxoid), harvested and then suspended in
Our previous investigations demonstrated that new bacilli sterile water adjusted to a 0.5 McFarland turbidity standard
isolated from shallow submarine hydrothermal vents of (bioMérieux), corresponding to 1.5 9 108 CFU ml-1. Sus-
Panarea Island (Italy) are able to produce EPSs useful in pensions were inoculated in triplicate onto plates of Mueller-
biotechnological applications [21]. We recently described a Hinton agar using a cotton swab. After 24 h of incubation at
novel haloalkaliphilic, thermophilic Bacillus licheniformis 37 C, the diameter of inhibition zones was measured with a
strain T14 isolated from Panarea Island, easy to cultivate in precision caliper (Mitutoyo, Andover, UK).
short time in a wide range of temperature, pH, and salinity, Some antibiotics have been indifferently tested for both
able to produce a new EPS (EPS1-T14) in large quantity by Gram-negative and Gram-positive strains, despite their
using a relatively simple purification process [54]. EPS1- exclusive use towards one or the other type of bacteria.

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

Each bacterial strain was classified as resistant (R), inter- plates [10]. Overnight cultures in peptone water of each
mediate resistant (I), or susceptible (S) according to the strain were diluted up to the OD600 = 0.1 (equivalent to
breakpoints established by Clinical Laboratory Standards 1.5 9 108 bacteria ml-1), and wells of microtiter plates
Institute (CLSI, 2006) [9]. were filled (eight replicates) with aliquots of 100 ll each.
The plates were incubated at 37 C for 24 h without
Bacillus licheniformis Strain T14 Producer shaking for biofilm production, and nonadherent bacteria
of EPS1-T14 were removed by washing 5 times with distilled water, by
using a Plate Washer (Das Instrument, Italy). The adherent
Bacillus licheniformis strain T14 as producer of the novel bacteria (biofilms) were stained with 1 % crystal violet
EPS1-T14 has been previously described [54]. Briefly, the solution (w/v) for 45 min [43]. Excess stain was removed
strain was isolated from a thermal fluid sample collected at by aspiration, and the plates were washed (5 times) and air
8 m depth from Bottaro vent, located off the eastern coast of dried (for 45 min). The stained biofilms were solubilized
Panarea Island (Eolian Islands, Italy). Characteristics of the with ethanol 99 %. Biofilm mass, estimated by the level of
emitted fluid were temperature 50 C, pH 5.42, and conduc- the crystal violet present in the destaining solution, was
tivity 42.90 mS m-1. The strain grew aerobically from 25 to spectrophotometrically (OD = 600 nm) determined using
60 C and its optimal temperature occurred at 50 C. The pH a microtiter plate reader (Multiskan GO, Thermo Scientific,
range for growth was 4–10, with optimum at pH 8. Strain T14 USA). The average OD from the control wells was sub-
grew in a range 2–10 % NaCl with optimal growth at 5 %. The tracted from the OD of all tested wells.
exopolysaccharide was obtained at the beginning of the sta-
tionary growth phase of the strain in the minimal medium Antibiofilm Assays
SWY (yeast extract, 0.1 %, sucrose 5 %, in seawater) after
48 h of incubation in bioreactor under optimal growth con- Pre-screening: Antibiofilm Effects of Strain T14
dition (temperature 50 C, pH 8, and NaCl 5 %). The purified Supernatants
fraction EPS1-T14 contained fructose/fucose/glucose in a
relative proportion of 1.0:0.75:0.28 and galactosamine and In a preliminary step, supernatants of B. licheniformis
mannose as contaminants. It possessed a molecular weight of strain T14 from two cultural media, differing in nutritional
1000 kDa, displaying a trisaccharide unit constituted by sources, were evaluated for their capacity to inhibit biofilm
sugars with a b-manno-pyranosidic configuration. formation of the clinical strains. Strain T14 was inoculated
into Bacto Marine Broth 2216 (Difco Laboratories, Detroit,
Measurement of the Reduction of Surface Tension MI) (MB) and into SWY containing yeast extract (0.1 %,
and the Emulsifying Activity of EPS1-T14 w/v) plus sucrose 5 % as carbon source. The cultures were
incubated under strain T14 optimal growth conditions
The reduction of the surface tension and the emulsifier (temperature of 50 C, pH 8, and NaCl 5 %) for 48 h and
activity (E24) of the EPS1-T14 were tested. The surface then centrifuged at 10,000g for 30 min to separate the cell
tension of the EPS1-T14 solution (0.5 %, w/v) was deter- pellets from media. To remove all bacterial cells, super-
mined with a digital tensiometer K10T (Krüss, Hamburg, natants were filtered through a 0.2-lm-pore-size membrane
Germany) by using Wilhelmy Plate method, which measures (Sartorius), and to ensure that no cells were present in the
the weight of the liquid drawn by a plate when a plate is lifted filtrates, 100 ll was spread onto plates of Bacto Marine
from or through the surface of liquid. The weight of the liquid Agar 2216 (Difco Laboratories, Detroit, MI).
is proportional to the surface tension of the liquid [26]. In order to enhance the antibiofilm activity, different
To test the emulsifying activity, 2 ml of an EPS1-T14 concentrations of cell-free supernatants (CFS) were used,
aqueous solution (0.5 %, w/v) was mixed with an equal including also the addition of tenfold concentrated super-
volume of kerosene in a glass tube (10 cm high and 1 cm natants (CFSC).
in diameter) and stirred in the vortex at high speed for To obtain CFSC, an aliquot (10 ml) of each supernatant
2 min. The emulsion and aqueous layers were measured was lyophilized and dissolved in 1/10 of sterile distilled
after 24 h, and emulsification index (E24) was calculated by water (resulting in 10 9 concentration). In a 96-well
the following formula: E24 = (Volume of the emulsion microtiter plate containing 100 ll of each strain culture
layer/Total volume) 9 100. (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
Biofilm Production and 16 % (v/v). The reduction of biofilm formation of each
clinical strain was expressed as antibiofilm activity (%), by
To evaluate the biofilm formation by the clinical strains, applying the following formula: (ODcontrol - ODsample/
the assay was carried out in 96-well polystyrene microtiter ODcontrol) 9 100. Each data point was averaged from eight

A. Spanò et al.: In Vitro Antibiofilm Activity of an Exopolysaccharide from the Marine… 521

replicated wells, and the standard deviation (SD) was Tryptone Soya Agar (Difco). Aliquots of 20 ll of CFS and
calculated. CFSC were applied to sterile filter paper discs (bioMérieux,
6 mm in diameter). The disks were placed onto the inocu-
Screening: Antibiofilm Activity of EPS1-T14 lated plates. Plates were incubated overnight at 37 C. The
diameter of complete inhibition zones was measured, and
To evaluate the ability to inhibit bacterial biofilm formation means and standard deviations were calculated.
by clinical strains, overnight cultures (100 ll) of each strain The antibacterial effects of EPS1-T14 on the clinical
were added to a 96-well microtiter plate together with EPS1- strains cultures were observed by growth curve analysis.
T14 at different final concentrations (25, 50, 100, 200, and Microtiter plates were filled (eight replicates) with over-
400 lg ml-1). The biofilm formation was assessed spec- night cultures in peptone water (100 ll) of each clinical
trophotometrically in the presence or without EPS1-T14. strain (OD600 = 0.1), with the addition of EPS1-T14 at a
The reduction of biofilm formation of each clinical strain final concentration of 400 lg ml-1. The plates were incu-
was expressed as antibiofilm activity (%), calculated as bated at 37 C for 24 h without shaking. Bacterial cultures
follows: (ODcontrol - ODsample/ODcontrol) 9 100. Each data without the addition of the EPS were used as control.
point was averaged from eight replicated wells and the SD OD600 values were recorded for up to 24 h at 1-h intervals.
was calculated.

Confocal Microscopic Observation of Biofilm Results

To directly observe the multicellular structures into the bio- Clinical Strains: Antibiotic Resistance
films, with or without the addition of EPS1-T14, the Confocal
Laser Scanning Microscopy (CLSM), with specific fluores- Antibiotic sensitivity/resistance pattern of the clinical
cent markers, was used [6]. As previously described, aliquots strains are reported in Table 1. Strains were resistant to the
of overnight cultures in peptone water (adjusted to major part of the tested antibiotics, and P. aeruginosa
OD600 = 0.1) were distributed in chamber slides (Nunc Inc., strain was susceptible only to fosfomycin.
Naperville, Illinois) for CLSM observations. After incubation
at 37 C for 24 h, the bacteria that remained in suspension Effects of B. licheniformis T14 Supernatants
were eliminated and the remaining adherent cells on the slide, on Biofilms Formation
after washing with Phosphate-Buffered Saline (PBS), were
fixed by heat and finally stained with 20 lg ml-1 of propid- A preliminary assessment of the antibiofilm activity was
ium iodide (PI) (Sigma), a nucleic acids’ intercalating and performed by the microtiter plate assay using the cell-free
fluorescent agent. supernatants obtained from strain T14 grown into different
The slides were incubated in the dark at 30 C for 5 min culture conditions. The supernatant obtained by using
to enable the fluorescent labelling of the bacteria. The flu- medium MB did not show any antibiofilm activity (data not
orescence excitation maximum is 535 nm and the emission shown), while the cell-free supernatants derived from
maximum is 617 nm. Observations were performed by medium SWY (plus 5 % of sucrose) (CFS and CFSC) were
CLSM using a TCS SP2 microscope (Leica Microsystems effective against all the tested strains. These results indi-
Heidelberg, Mannheim, Germany), equipped with Ar/Kr cated that, differently from the composition of medium
laser, and coupled to a microscope (Leica DMIRB). MB, the high content of sucrose as a unique carbon source
in medium SWY greatly influenced the production from
Antibacterial Activity strain T14 of biomolecules able to reduce the biofilm for-
mation from the tested strains.
To investigate whether the inhibitory effects of supernatants The antibiofilm activity of the cell-free supernatants was
(CFS and CFSC) from strain T14 and of EPS1-T14 on concentration dependent and differed among the tested
biofilm formation by clinical strains might be due to the bacteria (Table 2). CFS at a concentration of 2 % showed a
direct growth inhibition, antibacterial activity was evaluated. strong antibiofilm activity (C50 % of biofilm reduction)
Supernatants from strain T14 were tested by using the agar only against E. coli. At the highest concentration of CFS
diffusion method, as accepted by the National Committee for (16 %), the percentage of biofilm reduction was strong for
Clinical Laboratory Standards (NCCLS 2000). Each clinical all the target strains, with the only exception for K. pneu-
strain was overnight grown into Tryptone Soya Broth (Difco, moniae (42 %), and it reached the maximum value (69 %)
Becton–Dickinson and Company, USA) for 24 h at 37 C. for E. coli (Table 2).
Suspensions of each strain (equivalent to a 0.5 McFarland An enhanced biofilm inhibition activity was observed by
standard) were inoculated in triplicate onto plates of adding the tenfold concentrated supernatants (CFSC)

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

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.

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

Control EPS1-T14 (400 µg ml-1) 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
E. coli 463

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.
K. pneumoniae 2659


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
P. aeruginosa 445

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
S. aureus 210

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
Fig. 2 Confocal laser imagines (9400) of biofilm formed by E. coli are very few and include clavan [56], fucogel [45], and
463, K. pneumoniae 2659, P. aeruginosa 445, and S. aureus 210 in fucopol [19]. Their functional characteristics, such as
the absence (control) or presence of EPS1-T14 (400 lg ml-1) thickening, emulsion stabilizing, film forming, and floc-
culating capacities, have an increased interest in many
T14 at 400 lg ml-1 was also visualized onto glass surfaces biotechnological applications [20, 50, 56]. Particular
by confocal laser scanning microscopic images (Fig. 2). attention was paid towards the medical and pharmaceutical
To evaluate whether the antibiofilm effects were related applications of fucose-containing polysaccharides, as
to reduction of growth rate of the target strains, growth anticarcinogenic and antiinflammatory agents, useful in the
curves were measured in the presence and absence of treatment of rheumatoid arthritis, in age-related patholo-
EPS1-T14. The presence of EPS1-T14 at the highest con- gies accompanied by tissue loss, in acceleration of wound
centration used (400 lg ml-1) did not influence the growth healing and as hydrating and antiaging additives [8, 46].
rates of the tested clinical strains (Fig. 3), indicating that However, no antibiofilm activities of clavan, fucogel, and
the biopolymer have no antibacterial activity. These results fucopol have been reported until now.
showed that bacterial biofilm inhibition did not due to a Recently, we reported that EPS1-T14 is able to stimulate the
direct reduction of growth rate of the target strains, but to immune response and thus contribute to the antiviral immune
an inhibition of their biofilm formation. 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-
Biosurfactant Activities of EPS1-T14 positive, multiresistant clinical strains.
Sayem et al. [51] reported that cell-free supernatants
A significant reduction in surface tension (from 65.2 ± 3 obtained from the sponge associated B. licheniformis SP1
to 42.4 ± 3 mN m-1) was recorded in the presence of showed inhibition effects on biofilm formation by E. coli
EPS1-T14 (0.5 %, w/v). PHL628 and P. fluorescence (about 89 and 80 % of

A. Spanò et al.: In Vitro Antibiofilm Activity of an Exopolysaccharide from the Marine… 525

E. coli 463 K. pneumoniae 2659

1.2 1.2

1.0 1.0

0.8 0.8

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


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

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 prospective in medical and nonmedical applications, as
outer membrane cells. antiadhesive drug.
The presence of fructose and fucose as major compo-
Compliance with Ethical Standards
nents may provide additional biological properties to
EPS1-T14 compared to those composed of more common Conflict of interest The authors declare no conflict of interest.
sugar monomers. Fucose and fructose are inhibitors of the
surface lectins (proteins designed as fimbriae or pili) of
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