Hindering the formation and promoting the dispersion of medical biofilms: non-lethal effects of seagrass extracts

Hindering the formation and promoting the dispersion of medical biofilms: non-lethal effects of... Background: Biofilms have great significance in healthcare-associated infections owing to their inherent tolerance and resistance to antimicrobial therapies. New approaches to prevent and treat unwanted biofilms are urgently required. To this end, three seagrass species (Enhalus acoroides, Halophila ovalis and Halodule pinifolia) collected in Vietnam and in India were investigated for their effects in mediating non-lethal interactions on sessile bacterial (Escherichia coli) and fungal (Candida albicans) cultures. The present study was focused on anti-biofilm activities of seagrass extracts, without killing cells. Methods: Methanolic extracts were characterized, and major compounds were identified by MS/MS analysis. The antibiofilm properties of the seagrass extracts were tested at sub-lethal concentrations by using microtiter plate adhesion assay. The performance of the most promising extract was further investigated in elegant bioreactors to reproduce mature biofilms both at the solid/liquid and the solid/air interfaces. Dispersion and bioluminescent assays were carried out to decipher the mode of action of the bioactive extract. Results: It was shown that up to 100 ppm of crude extracts did not adversely affect microbial growth, nor do they act as a carbon and energy source for the selected microorganisms. Seagrass extracts appear to be more effective in deterring microbial adhesion on hydrophobic surfaces than on hydrophilic. The results revealed that non-lethal concentrations of E. acoroides leaf extract: i) reduce bacterial and fungal coverage by 60.9 and 73.9%, respectively; ii) affect bacterial biofilm maturation and promote dispersion, up to 70%, in fungal biofilm; iii) increase luminescence in Vibrio harveyi by 25.8%. The characterization of methanolic extracts showed the unique profile of the E. acoroides leaf extract. Conclusions: E. acoroides leaf extract proved to be the most promising extract among those tested. Indeed, the selected non-lethal concentrations of E. acoroides leaf extract were found to exert an antibiofilm effect on C. albicans and E. coli biofilm in the first phase of biofilm genesis, opening up the possibility of developing preventive strategies to hinder the adhesion of microbial cells to surfaces. The leaf extract also affected the dispersion and maturation steps in C. albicans and E. coli respectively, suggesting an important role in cell signaling processes. Keywords: Seagrass extracts, Non-lethal concentrations, Antibiofilm activity, Escherichia coli, Candida albicans * Correspondence: federica.villa@unimi.it Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, Università degli Studi di Milano, via Celoria 2, 20133 Milan, Italy Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 2 of 17 Background extracts, including antibacterial, antifungal, antialgal, anti- The ability of microorganisms to colonize surfaces and oxidant, anti-inflammatory, insecticidal, antimalarial and develop into highly organized communities enclosed in a vasoprotective properties, have been reported [26–28]. self-produced polymeric matrix is the predominant Thus, the well described properties of seagrasses ex- growth modality in both nature and artificial systems. tracts offer a promising framework for investigating novel Such lifestyle is called biofilm and it is characterized by antibiofilm activities at non-lethal concentrations. alterations in microbial phenotypes with respect to growth The present study explores, for the first time, the effect rates and gene transcriptions [1–3]. of extracts from different seagrasses (namely, leaves and Biofilms have great significance for public health, roots from Enhalus acoroides Rich. ex Steud., Hydrochari- representing 65–80% of microbial diseases currently taceae, leaves of Halophila ovalis (R.Br.) Hook.f., Hydro- treated by physicians in the developed world [4, 5]. The charitaceae, and leaves of Halodule pinifolia (Miki) presence of indwelling medical devices further increases Hartog, Cymodaceaceae) in mediating non-lethal interac- the risk for biofilm formation and subsequent infection tions on sessile Candida albicans and Escherichia coli [6]. The bacterium Escherichia coli and the polymorphic cultures, selected as model systems for fungal and bacter- fungus Candida albicans are among the most frequent ial biofilm infections, respectively. The work focuses on cause of bloodstream infections, and the predominant investigating the antibiofilm performance of seagrass ex- microorganisms isolated from infected medical devices tracts at sub-inhibitory concentrations, studying how they [7, 8]. These biofilms, as any other biofilm, exhibit affect biofilm functional traits (such as adhesion, biofilm dramatically decreased susceptibility to antimicrobial maturation, dispersal and quorum sensing), and induce agents and resistant to the host immune clearance, which cellular responses other than those associated with anti- increases the difficulties for the clinical treatment of infec- microbial activities. tions [9–11]. Furthermore, the antimicrobial arena is ex- periencing a shortage of lead compounds, and growing Methods negative consumer perception against synthetic products Plant material and extraction has led to the search for more natural solutions [12]. Three species of seagrasses (leaves and roots from In this context, it has been reported that plant-derived Enhalus acoroides Rich. ex Steud., Hydrocharitaceae, extracts exhibit good antibiofilm properties against a leaves of Halophila ovalis (R.Br.) Hook.f., Hydrocharita- range of microorganisms [13–15]. However, in the past, ceae, and leaves of Halodule pinifolia (Miki) Hartog, these extracts were mainly screened by focusing on their Cymodaceaceae) were collected in Vietnam and India lethal effects [16–18] disregarding their activity at and air-dried in a dark place (Table 1). Enhalus acor- non-lethal concentrations. At these concentrations, oides and Halophila ovalis were collected and identified plant-derived extracts may reveal elegant mechanisms by Xuan-Vy Nguyen, Department of Marine Botany, to sabotage the sessile lifestyle, manipulating the ex- Institute of Oceanography, Vietnam Academy of Science pression of stage-specific biofilm phenotypes [19]. For and Technology, Nha Trang City, Vietnam, based on mor- instance, by affecting the cellular ability to attach to phological characters and controlled by ITS molecular surfaces and by mystifying intercellular signals, the marker analysis [29]. Specimens of Enhalus acoroides are biofilm cascade might be hampered. Thus, non-lethal stored in the herbarium of the Institute of Botany, concentrations of plant-derived extracts can inspire in- Hannover, Germany (Specimen number: EA20130301). novative, eco-friendly and safe strategies aim at treating Halodule pinifolia was collected by Jutta Papenbrock and deleterious biofilms. Interfering with specific key steps further identified by Thirunavakkarasu Thangaradjou, that orchestrate biofilm genesis might offer new ways Centre of Advanced Study in Marine Biology, Annamalai to disarm microorganisms without killing them, side- University, Parangipettai, Tamilnadu, India, based on mor- stepping drug resistance [4]. phological characters and controlled by ITS molecular Seagrasses, which belong to the halophytes, represent a marker analysis [30]. Specimens are stored in the functional group of underwater marine flowering plants herbarium of the Annamalai University, Parangipettai, that have developed several strategies to survive and re- Tamilnadu, India. produce in environments where the salt concentration is The plants were separated into different organs (leaves around 200 mM NaCl or more [20]. As these plants grow and roots), and samples were cooled with liquid nitrogen in very high saline conditions, it is predicted that they and ground to a fine powder using a bead mill (Retsch), could possess rare and new activities not reported for their three times for 10 s at a frequency of 30/s. The samples terrestrial relatives [21, 22]. Indeed, metabolomic studies were stored at − 80 °C prior to analysis. Crude extracts have shown that increased salinity leads to changes in were obtained using 80% methanol (MeOH) as solvent. conserved and divergent metabolic responses in halo- Around 50 mg of powdered seagrass material was weighed phytes [23–25]. Moreover, interesting activities of seagrass in a reaction tube and extracted with 800 μl80% MeOH De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 3 of 17 Table 1 Seagrass species and information about collection sites Species Plant organ Collection site GPS Collection date Enhalus acoroides Leaf Nha Trang Bay, Vietnam 109.209208°E 19.04.2011 12.158073°N Enhalus acoroides Root Nha Trang Bay, Vietnam 109.209208°E 19.04.2011 12.158073°N Halophila ovalis Leaf Nha Trang Bay, Vietnam 109.209208°E 19.04.2011 12.158073°N Halodule pinifolia Leaf Chilika Lagoon, India 85.418015°E 16.02.2010 19.775105°N for 10 min with regular shaking. Then the extract was Quantification of total phenolic contents (TPC) centrifuged for 5 min at 18000 x g and the supernatant To measure the total phenolic acid content, a modified transferred into a new reaction tube. These steps were re- protocol after Dewanto et al. [32] was used with the peated three times with 400 μl 80% MeOH each. The su- same extracts described above. 96-well microtiter plate pernatants were collected in the same reaction tube and were filled with 100 μlH O each. From each sample, stored at − 20 °C. Phosphate buffered saline (PBS, 0.01 M 10 μl were added; seagrass extracts were diluted 1:2. A phosphate buffer, 0.0027 M potassium chloride 0.137 M, gallic acid calibration curve with the following concen- sodium chloride, Fisher Scientific) was used to obtain sev- tration was used: 0, 5, 10, 25, 50, 75, 100, 125 and eral concentrations of each crude extract: 100, 10, 1, 0.1, 250 μg/ml. Next, 100 μlNa CO 7% were added and the 2 3 0.01 and 0.001 mg/l. plate was incubated for 100 min in the dark. The ab- sorption was measured at 765 nm in a microplate reader. With the slope of the gallic acid calibration Microbial strains and growth media curve, the concentration of phenolic acids was calculated The microbial strains Candida albicans SC5314 (ATCC in mg gallic acid equivalent. MYA-2876) and Escherichia coli K-12 wild-type strain (ATCC 25404) were selected as model systems for fun- Determination if the oxygen radical absorbance capacity gal and bacterial biofilms respectively. C. albicans and (ORAC) E. coli strains were stored at − 80 °C in suspensions The analysis of the oxygen radical absorbance capacity containing 50% glycerol and 2% peptone, and were rou- (ORAC) was conducted according to a protocol based tinely grown in amino acid-free yeast nitrogen base (YNB, on Huang et al. (2002) [33] and Gillespie et al. [34] Sigma-Aldrich) supplemented with 0.5% glucose (YNBG, with the same extracts. A black 96-well microtiter was Conda) and Luria-Bertani broth (LB, Sigma-Aldrich), used and the wells were filled with 120 μl fluorescein respectively, for 16 h at 30 °C. (112 nM) in phosphate buffer (75 mM, pH 7.4). Of each sample and the standard curve, 20 μl were added Quantification of total flavonoid contents (TFC) in each well. The standard curve of 6-hydroxy-2, The total flavonoid content of the seagrass extracts was 5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) measured in 96-well plate according to a modified was prepared in phosphate buffer with the following protocol from Dudonné et al. [31]. The wells were filled concentrations: 6.25, 12.5, 25 and 50 μM. Seagrass with 150 μlH O each. Dilutions of the methanolic sea- extracts were diluted 1:200 with methanol 80%. The grass extracts (1:2) were prepared and 25 μl of sample microtiter plate was incubated for 15 min at 37 °C. The were filled in one well, with four replicates. A calibration fluorescence was then measured at 485/520 nm as time curve with catechin hydrate with the following concen- point zero. Next, 80 μlof 2,2′-azobis(2-amidino-propane) trations was prepared in 80% MeOH: 0, 10, 25, 50, 100, dihydrochloride (62 mM) were added and the fluores- 125, 250 and 400 μg/ml. The calibration curve was cence was measured every minute for 80 min. The ORAC placed on the plate in triplicate. In the next step, 10 μl value was calculated as the difference between time point NaNO 3.75% were added into each well and incubated zero and 80 min and quantified with the Trolox standard for 6 min. Afterwards, 15 μl of AlCl 10% were added curve. and incubated for 10 min. In the last step, 50 μlof NaOH 1 M were added and the absorption was mea- LC-MS analysis sured at 510 nm in a microplate reader (Biotek, Winoo- LC-MS analysis was performed on a Shimadzu HPLC ski, USA). The slope of the calibration curve was used to system (controller CBM-20A, two pumps LC-20 AD, a calculate the total flavonoid content in mg catechin column oven CTO-20 AC and a photo diode array equivalent. detector SPD-M20A; Shimadzu, Darmstadt, Germany) De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 4 of 17 coupled to a Triple Tof 4600 mass spectrometer (AB 60 min for 30 h in wells inoculated with 45 μl (3% vol/vol) Sciex, Canby, USA). The separation of extracted com- of an overnight culture (approximately 10 cells/ml). The pounds was realised on a Knauer Vertex Plus column negative control was represented by PBS supplemented (250 × 4 mm, 5 μm particle size, packing material with 45 μl (3% vol/vol) of the overnight culture. The ProntoSIL 120–5 C18-H) with precolumn (Knauer, polynomial Gompertz model [37] was used to fit the Berlin, Germany). The column oven temperature was growth curves to calculate the maximum specific growth set to 30 °C and 25 μl of undiluted methanolic seagrass rate (A /min), using GraphPad Prism software (version extract prepared as described above was injected. The 5.0, San Diego, CA, USA). Five biological replicates of solvent flow rate was 0.8 ml/min. In this time, a gradi- each treatment were performed. ent was run from 10 to 90% B from minute 0 to 35, 2min of90% B, switch to 10%Bin1min andsubse- Microplate-based biofilm assay quent equilibration at 10% B for 2 min. Solvent A The antibiofilm activity of seagrass extracts was assessed (water) and B (methanol) were both supplemented with quantitatively as previously reported by Villa et al. [38]. 2 mM ammonium acetate and 0.01% acetic acid. Mass Briefly, 200 μl of PBS containing 10 cells/ml supple- spectra were monitored between 100 and 800 Da in mented with 0 (positive control), 100, 10, 1, 0.1, 0.01, negative ionisation mode. In addition, MS/MS spectra and 0.001 mg/l of each crude extract were placed in were generated with a collision energy of − 30 eV and hydrophobic and hydrophilic 96-well polystyrene-based measured between 50 and 800 Da. Spectra for the most microtiter plates (Thermo Fisher Scientific). After an prominent peaks were compared to database entries in incubation time of 24 h at 20 °C, C. albicans and E. coli MassBank [35]and ReSpect [36] for identification. planktonic cells were removed and adhered cells were stained using 0.1 mg/ml of Fluorescent Brightener 28 vital Planktonic growth in the presence of seagrass extracts as dye (Sigma-Aldrich) or 4′, 6-diamidino-2-phenylindole the sole source of carbon and energy (DAPI, Sigma-Aldrich) in PBS, respectively. After 20 min The ability of C. albicans and E. coli planktonic cells to staining in the dark at room temperature the microtiter grow in the presence of each extract as the sole carbon plates were washed twice with 200 μl PBS and the fluores- and energy source was tested using YNB and M9 cence intensity due to adhered cells was measured using a (Sigma-Aldrich) mineral medium, respectively, supple- fluorescence microplate reader (TECAN, Manneford, mented with the highest working extract concentration: Switzerland) at excitation wavelength of 335 nm and emis- 100 mg/l. Then a 100 μl mix of mineral medium to- sion wavelength of 433 nm. A standard curve of fluores- gether with 45 μl (3% v/v) of the overnight culture (final cence intensity versus cell number was determined and concentration 10 cells/ml) and the highest concentra- used to quantify the antibiofilm performance of the crude tion of each marine plant extract were used to fill each extracts. Percentage reduction with respect to the positive well of 96-well plates (Thermo Fisher Scientific) and in- control is calculated as (treated data –control data) × 100 cubated for 48 h at 30 °C. A medium complemented / control data. Cattò et al. [39] proposed the following with cells and glucose (5 g/l), and medium without cells, anti-adhesion ranges computing the percentage reduction were used as positive and negative controls, respectively. in comparison to the negative control: ≤20% without Microbial growth was monitored using the PowerWave anti-adhesion activity; between 20 and 30% and 30 and XS2 microplate reader (Biotek) measuring the absorb- 40% low anti-adhesion activity and with moderate ance at 600 nm (A ) every 10 min. Six biological repli- anti-adhesion activity respectively; ≥40% adhered cells cates of each treatment were performed. The obtained with excellent anti-adhesion activity. Five biological repli- data were normalized to the negative control and re- cates were performed for each condition and a percentage ported as the mean of these. reduction in comparison to the negative control was calculated as (treated data – positive control data) × 100/ Growth inhibition assay in the presence of seagrass positive control data. The experiment was repeated three extracts times. The ability of the seagrass extracts to inhibit the plank- tonic growth of the selected microorganisms was investi- Biofilm growth at the solid/liquid interface gated. For this, C. albicans and E. coli were grown YNBG The most promising plant extracts were screened for and LB broth respectively without (positive control) and their effects on biofilm development. C. albicans biofilm with the highest working concentrations (10 and 100 mg/ was grown in the CDC biofilm reactor (Biosurface l) in 96-well plates (Thermo Fisher Scientific). Growth Technologies, Bozeman, MT, USA) as previously de- curves at 30 °C were generated using Infinite® F200 PRO scribed by Villa et al. [40]. Briefly, two bioreactors hosting microplate reader (TECAN, Mannedorf, Switzerland) by 24 polycarbonate coupons (to simulate a hydrophobic measuring the optical density at 600 nm (OD ) every surface) were filled with YNBG and 1 ml of overnight 600 De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 5 of 17 planktonic culture (approximately 10 cells/ml) and, in (plate well). Biofilm formation was performed at 37 °C in one of them, 0.01 mg/l of E. acoroides leaf extract was aerobic conditions for 16 h. At different time points (0, 4, added. Bioreactors were maintained under static condi- 6, 8, 16 h) some membranes were removed, biofilm was tions (no flow) for 24 h under mild stirring at 37 °C, pro- scraped off using a sterile loop, put inside a tube contain- moting fungal adhesion to the surface of the removable ing 1 ml of PBS and then homogenized twice using a polycarbonate coupons. After that, the dynamic phase was homogenizer (IKA T10 basic Ultra-Turrax – Cole-Parmer initiated and diluted YNGB was fluxed for 48 h at flow Instrument Company). Then serial dilutions were pre- rate of 250 ml/h. Biofilm growth in the absence (positive pared and 10 μl were inoculated in petri dishes containing control) and presence of the extract was evaluated by LB with agar following the drop counting method. After quantification of the biomass. At different time steps (24, 12 h at 37 °C, E. coli colonies were counted and the bio- 48 and 72 h) some polycarbonate coupons were collected mass was quantified. This assay was assessed under three in aseptic conditions and resuspended in 3 ml of PBS experimental conditions: i) treatment 1: growth in contact each. Subsequently, serial dilutions were carried out, and with 1 ml of LB with 10 mg/l of E. acoroides leaf extract 10 μl were inoculated in petri dishes containing Tryptic for 16 h; ii) treatment 2: overnight culture grown with Soy Broth medium (TSB, Sigma-Aldrich) complemented 10 mg/l of E. acoroides leaf extract, and then growth in with agar (Merck) following the drop counting method. contact with 1 ml of LB for 16 h; iii) treatment 3: over- After 12 h at 30 °C, C. albicans colonies were counted and night culture grown with 10 mg/l of E. acoroides leaf ex- the data obtained were normalized to the coupon area, tract, and then growth in contact with 1 ml of LB with and means were reported. The same protocol was used to 10 mg/l of E. acoroides leaf extract for 16 h. In the positive obtain mature biofilm of E. coli, using LB as a medium, control, the microorganisms grew in 1 ml LB inside a and evaluating 10 mg/l of E. acoroides leaf extract. Each basolateral well for 16 h without the extract. The data ob- experiment was repeated three times. tained were divided by the area of the membrane, and the means were reported. The experiment was repeated three Biofilm dispersion assay times. Mature C. albicans biofilm was grown in the CDC reactor in the absence (positive control) and presence B2ioluminescence assay using Vibrio harveyi and of 0.01 mg/l of E. acoroides leaf extract as reported Two hundred μl of autoinducer bioassay (AB) mineral below. As previously described by Cattò et al. [41], after medium (0.3 M NaCl, 0.05 M MgSO ,0.5%casein 72 h polycarbonate coupons were collected, immersed in hydrolysate, 10 μMKH PO ,1 μM L-arginine, 50% 2 4 27 ml of PBS for one minute at room temperature, serial glycerol, 0.01 μg/ml riboflavin, 1 μg/ml thiamine. pH 7. dilutions were carried out and 10 μl were inoculated in Sigma-Aldrich) containing 10% (V/V) of a tenfold dilu- petri dishes containing TSB supplemented with agar tion of an overnight culture of Vibrio harveyi BB170 (Merck) following the drop counting method. After 12 h (ATCC BAA-1117) grown in AB medium were supple- at 30 °C, C. albicans colonies were counted and the per- mented with 10 mg/l of E. acoroides leaf extract re- centage of biofilm dispersion was calculated as (number spectively, and were placed in hydrophobic 96-well of viable cells from bulk PBS × 100) / (number of viable polystyrene-based microtiter plates (Thermo Fisher cells from bulk PBS + number of viable cells from the Scientific) with transparent bottom. The positive control coupon biofilm) and means were reported. Three bio- was an AB mineral medium supplemented with 10% (V/ logical replicates were performed for each treatment and V) tenfold dilution of the overnight culture. Absorbance six technical replicates were performed for each experi- (OD ) and luminescence were measured using a mi- 600nm ment. The experiment was performed three times. croplate reader (VICTOR™X, Perkin Elmer, USA) every 8 h for 24 h, incubating the microtiter plate at 30 °C Biofilm growth at the solid/air interface during the experiment. The data obtained were normal- E. coli biofilm was grown on a sterile polycarbonate mem- ized to the number of viable cells, divided by the area of brane (PC, Whatman Nucleopore, diameter 2.5 cm, pore the membrane, and the means reported. The experiment diameter 0.2 μm) as previously described by Garuglieri et was repeated three times. al. [42]. Briefly, 0.05 ml of an overnight culture (approxi- mately 10 cells/ml) were inoculated at the center of a sterile polycarbonate membrane and, when the inoculum Statistical analysis was completely dried, the membrane was carefully put in- To evaluate statistically significant differences among side a transwell structure (ThinCert™ Cell Culture Inserts samples, analysis of variance (ANOVA) via MATLAB with translucent PET membrane – Greiner bio-one) inlaid software (Version 7.0, The MathWorks Inc., Natick, in a 6 well culture plate (Greiner bio-one). One ml of LB USA) was applied. Tukey’s honestly significant different medium was inoculated in the basolateral compartment test (HSD) was applied for pairwise comparison to De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 6 of 17 establish the significance of the data. Statistically sig- of E. acoroides also contained two kaempferol-based flavo- nificant results were represented by P values ≤0.05. noles and luteolin and also a procyanidin and a flavanole (epicatechin). In H. ovalis three flavonoids and one phen- Results olic acid was found. H. pinifolia contained several flavo- Seagrass extracts contain phenolic compounds and show noles, either based on kaempferol or quercetin and also antioxidant capacities epicatechin. The methanolic extracts from the seagrass material contained phenolic acids as well as flavonoids (Fig. 1a-b). Seagrass extracts are not used as carbon and energy The content of phenols and flavonoids was highest in H. source by C. albicans and E. coli and do not affect their pinifolia leaf extracts with 18.0 ± 0.25 and 14.3 ± 0.25 mg/ planktonic growth g dry mass (DM), respectively. In E. acoroides, the root C. albicans and E. coli planktonic cells grown only in the material showed higher amounts of total flavonoids and presence of medium supplemented with glucose were phenols than the leaf material. For all seagrass species, the used as the positive control of the experiment (Fig. 3). content of phenolic acids was higher than the flavonoid Note that the mineral medium supplemented with the content with respect to the DM. highest concentration of tested plant extracts did not Methanolic extracts from the four seagrass species promote the growth of the selected microorganisms. were analyzed for their antioxidant capacity (Fig. 1c). The response of the planktonic growth of the selected All tested extracts had the ability to absorb oxygen rad- microorganisms in the presence of the seagrass extracts at icals. H. pinifolia showed the highest activity with 97.7 the highest concentrations (10 and 100 mg/l) is reported ± 2.7 mg Trolox equivalents (TE)/g DM. E. acoroides in Figs. 4 and 5. C. albicans and E. coli growth rates (table and H. ovalis leaf extracts showed similar antioxidant in Figs. 4 and 5) showed that there are no statistically sig- capacities with 70.2 ± 4.1 and 72.5 ± 2.9 mg TE/g DM, nificant differences between the presence and the absence respectively. The root extract from E. acoroides displayed of the extracts obtained from every plant portion at any a lower ORAC value than the extract from the leaves tested concentration. Therefore, concentrations ≤100 mg/l (45.1 ± 3.2 mg TE/g DM). plant extract were used in the subsequent studies. LC-MS analysis of secondary metabolites E. acoroides leaf extract inhibits cell adhesion on a E. acoroides, H. ovalis and H. pinifolia show different hydrophobic surface compositions of secondary metabolites (Fig. 2). The The percentage reduction of the number of adhered cells identification of individual compounds in the methanolic of E. coli and C. albicans on hydrophilic and hydrophobic extracts was done via the comparison of MS/MS spectra surface in presence of non-lethal concentrations of sea- with database entries. The three seagrass species showed grass extracts is showed in Fig. 6. The results revealed that different profiles of secondary metabolites, in this case E. acoroides and H. ovalis were the most promising ex- mainly flavonoids and phenolic acids (Table 2). In E. acor- tracts for C. albicans, with excellent anti-adhesion activity, oides leaves, three flavonoles based on kaempferol were reducing fungal coverage up to 73.89 ± 1.01% and 68.37 ± found. In addition, two flavones (apigenin and luteolin), 2.49% at 0.01 and 1 mg/l, respectively. For E. coli,10 mg/l one phenolic acid (benzoic acid) and the saturated dicar- of E. acoroides leaf extract was found to be the concentra- boxylic acid azelaic acid were identified. The root extract tion with the highest reduction in cell adhesion (reduction Fig. 1 Crude methanolic extracts were analyzed for (a) Total phenols in mg gallic acid equivalent (GAE) per g dry mass (DM), (b) Total flavonoids in mg catechin equivalent (CE) per g DM, and (c) ORAC in mg Trolox equivalents (TE) per g DM. Data represent the mean ± SDs and different superscript letters indicate statistically significant differences (Tukey’s HSD, p ≤ 0.05) between the means of three independent measurements. (EAL = Enhalus acoroides leaf; EAR = Enhalus acoroides root; HPL = Halodule pinifolia leaf; HOL = Halophila ovalis leaf) De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 7 of 17 Fig. 2 Chromatograms from E. acoroides leaf extract (a), E. acoroides root extract (b), H. ovalis leaf extract (c) and H. pinifolia leaf extract (d) from minute 0–33. The relative intensity of mass between 100 and 800 Da is shown. Numbers indicate putatively identified substances in Table 2 of bacterial coverage by 60.86 ± 8.85%). Therefore, 0.01 mg/ were in contact with the extract during both overnight l and 10 mg/l E. acoroides leaf extract were chosen as the growth and biofilm formation (reduction of cellular best non-biocidal concentrations for C. albicans and E. coli growth, compared to the control, up to 48.64 ± 4.02%). respectively, and were used in the subsequent studies. This growth curve was characterized by two exponen- tial phases separated distinctly by an intermediate E. acoroides leaf extract does not impact on biofilm phase where the growth rate is very low. After that, at growth curves, but does induce biofilm dispersion in C. 16 h the number of viable cells was similar to the other albicans and interfere with AI2 treatments. A CDC reactor was used as the laboratory scale model The effects of 10 mg/l of E. acoroides leaf extract on system to grow a complex and mature C. albicans biofilm the cellular communication of V. harveyi were reported in the absence and presence of 0.01 mg/l E. acoroides leaf in Fig. 9. The results highlighted a significant increase in extract, the most effective concentration obtained from the relative luminescence emitted at time 8 h compared the adhesion assay. to the control (25.75 ± 7.49%). Results in Fig. 7a indicated a significant reduction in the number of viable cells adhered on coupon surfaces treated Discussion with the extract, compared to the untreated ones, after Biofilm resistance to antimicrobial agents is a major 24 h (reduction of fungal coverage up to 26.77 ± 9.01%). worldwide health care issue. Therefore, a successful re- Coupons collected after 48 and 72 h showed no significant duction of surface colonization can be a potential strat- differences between the treated biofilm and the control. egy for the management of unwanted biofilms, especially A significant increase in the number of dispersed on medical devices and work surfaces. cells in the treated biofilm (70 ± 6.83%) was observed In this context, the use of plant-derived extracts to (Fig. 7b). modulate biofilm genesis and dispersion may be a viable A colony biofilm assay was used to grow a complex alternative. The present study is the first report describ- and mature E. coli biofilm in the presence and absence ing the antibiofilm efficacy of non-lethal concentrations of 10 mg/l E. acoroides. Results in Fig. 8 showed no sig- of E. acoroides, H. pinifolia and H. ovalis methanol nificant reduction in the number of viable cells during extracts in counteracting microbial biofilms, highlighting biofilm formation on the membrane treated with the the possibility that the selected seagrass species act as an extract, compared to the untreated, after 18 h in all the extracellular signal mediating their biofilm activities. experimental conditions. Treatment 3 showed a growth E. coli and C. albicans were chosen as model systems rate slowdown in the interval 6–8, in which E. coli cells for bacterial and fungal infections, respectively. E. coli De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 8 of 17 Table 2 Individual compounds identified by comparison of MS/MS spectra with database entries in Enhalus acoroides leaf extract (A), E. acoroides root extract (B), Halophila ovalis leaf extract (C) and Halodule pinifolia leaf extract (D) No RT Mass MS/MS Name Accession Source A- E. acoroides leaf extract 1 2.5 343.03 201.02, 157.03, 59.01 n. i. –– 2 2.7 312.12 179.05, 132.06, 89.02 n. i. –– 3 3.3 367.1 277.07, 187.04, 157.03 n. i. –– 4 7.2 134.04 107.03, 92.02 Adenine PT200393 ReSpect 5 13.7 637.1 461.07, 285.04 Kaempferol-3-glucuronide, mod. PT209240 ReSpect 6 14.8 275.15 233.12, 119.05 n. i. –– 7 15.2 121.03 92.02, 77.03 Benzoic acid KO000321 MassBank 8 18.6 527.02 285.04, 241.00, 96.96 n. i. –– 9 20.1 511.05 269.04, 241.00, 96.96 n. i. –– 10 20.8 187.09 169.08, 125.09, 97.06 Azelaic acid KO000124 MassBank 11 21.3 447.09 285.04 Kaempferol-3-O-glucoside PS042209 ReSpect 12 22.5 461.07 285.04 Kaempferol-3-glucuronide PS092408 ReSpect 13 27.5 285.04 151.00, 133.03 Luteolin PS040410 ReSpect 14 29.5 269.04 225.05, 151.00, 117.03 Apigenin PT203930 ReSpect B- E. acoroides root extract 1 2.4 343.03 201.02, 157.03, 59.01 n. i. –– 2 2.7 312.12 179.05, 132.06, 89.02 n. i. –– 3 2.9 377.08 341.11, 179.05, 119.03, 89.02 Galactinol dihydrate, mod. PT211910 ReSpect 4 4.3 216.98 173.02, 156.98, 136.94, 59.01 n. i. –– 5 7.2 134.04 107.03, 92.02 Adenine PT200393 ReSpect 6 9.6 577.12 451.10, 425.08, 407.07, 289.07, 125.02 Procyanidin B2 PT204580 ReSpect 7 12.3 289.07 245.07, 203.07, 151.04, 109.03 +(−) Epicatechin PT204560 ReSpect 8 13.8 637.1 461.07, 285.04 Kaempferol-3-glucuronide, mod. PT209240 ReSpect 9 14.0 469.08 275.02, 193.05, 178.02, 149.06, 96.96 n. i. –– 10 14.8 275.15 233.12, 119.05 n. i. –– 11 15.3 121.03 92.02, 77.03 Benzoic acid KO000321 MassBank 12 20.8 187.09 169.08, 125.09, 97.06 Azelaic acid KO000124 MassBank 13 22.6 461.07 285.04 Kaempferol-3-glucuronide PS092408 ReSpect 14 24.1 299.05 284.03, 256.03, 133.03 Kaempferide PT204030 ReSpect 15 27.5 285.04 151.00, 133.03 Luteolin PS040410 ReSpect 16 29.5 269.04 225.05, 151.00, 117.03 Apigenin PT203930 ReSpect 17 31.2 329.23 229.14, 211.13, 171.10 n. i. –– C- H. ovalis leaf extract 1 2.4 343.03 201.02, 157.03, 59.01 n. i. –– 2 2.9 377.08 341.11, 179.05, 119.03, 89.02 Galactinol dihydrate, mod. PT211910 ReSpect 3 4.3 216.98 173.02, 156.98, 136.94, 59.01 n. i. –– 4 13.3 261.04 217.05, 189.05, 133.02 n. i. –– 5 15.5 121.03 92.02, 77.03 Benzoic acid KO000321 MassBank 6 16.3 306.17 288.16 n. i. –– 7 17.5 479.08 316,02 Myricetin-3-galactoside PS092809 ReSpect 8 19.5 463.09 301,03 Quercetin-3-O-beta-D-galactoside PS046509 ReSpect 9 20.8 187.09 169.08, 125.09, 97.06 Azelaic acid KO000124 MassBank De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 9 of 17 Table 2 Individual compounds identified by comparison of MS/MS spectra with database entries in Enhalus acoroides leaf extract (A), E. acoroides root extract (B), Halophila ovalis leaf extract (C) and Halodule pinifolia leaf extract (D) (Continued) No RT Mass MS/MS Name Accession Source 10 21.1 317.02 271.02, 149.02 n.i. –– 11 21.3 447.09 285.04 Kaempferol-3-O-glucoside PS042209 ReSpect 12 23.5 301.03 255.03, 165.02, 133.03 n.i. –– 13 24.1 299.05 284.03, 256.03, 133.03 Kaempferide PS040309 ReSpect 14 25.7 285.04 239.03, 185.06, 143.05, 117.03 Kaempferol PR040027 MassBank 15 27.5 285.04 285.04, 151.00,133.02 Luteolin PT204043 ReSpect 16 29.4 269.04 225.05, 151.00, 117.03 Apigenin PT203930 ReSpect D- H. pinifolia leaf extract 1 2.4 343.03 201.02, 157.03, 59.01 n. i. –– 2 2.9 377.08 341.11, 179.05, 119.03, 89.02 Galactinol dihydrate, mod. PT211910 ReSpect 3 4.3 216.98 173.02, 156.98, 136.94, 93.03, 59.01 n. i. –– 4 6.6 473.07 311.04, 293.03, 179.03, 149.01 n. i. –– 5 9.6 577.12 451.10, 425.08, 407.07, 289.07, 125.02 Procyanidin B2 PT204580 ReSpect 6 12.1 289.07 245.07, 203.07, 151.04, 109.03 +(−) Epicatechin PT204560 ReSpect 7 14.0 469.08 275.02, 193.05, 178.02, 149.06, 96.96 n. i. –– 8 19.1 641.17 473.13, 311.07, 167.03 n. i. –– 9 19.7 549.09 505.10, 463.09, 300.02, 271.02, 255.02 Quercetin-3-(6-malonyl)-glucoside PT209340 ReSpect 10 20.8 187.09 169.08, 125.09, 97.06 Azelaic acid KO000124 MassBank 11 21.1 505.09 463.08, 300.02, 271.02 Quercetin-3-O-beta-D-galactoside, mod. PT204650 ReSpect 12 21.8 463.08 300.03, 271.02 Quercetin-3-O-beta-D-galactoside PT204650 ReSpect 13 22.4 433.07 300.02, 271.02, 255.03, 179.00 Quercetin-3-arabinoside PT209320 ReSpect 14 23.4 447.09 284.03, 255.03, 227.03 Kaempferol-3-glucoside PT209270 ReSpect 15 24.6 417.08 284.03, 255.03, 227.03 Kaempferol-3-O-alpha-L-arabinoside PT209220 ReSpect 16 26.3 301.03 178.99, 151.00, 121.03, 107.01 Quercetin PT204090 ReSpect 17 27.4 285.04 199.03, 175.04, 151.00, 133.02 Luteolin PT204043 ReSpect 18 27.7 315.05 300.02, 271.02, 255.03 Isorhamnetin PM007432 ReSpect 19 29.5 269.04 225.05, 151.00, 117.03 Apigenin PT203930 ReSpect No = number of peak in Fig. 9, RT = retention time, Mass = mass of precursor ion, MS/MS = fragment spectra obtained at − 30 eV, Accession = accession number in database, Source = database used, n. i. = not identified, mod. = modified biofilms are found to be the major causative agent of antibacterial activity of H. ovalis and H. pinifolia extracts, many intestinal infections, for recurrent urinary tract in- obtained using different solvents, against different micro- fections, and it also responsible for indwelling medical bial strains, recording maximum antibacterial activity by device-related infectivity [43]. C. albicans is one of the the ethanol extract of H. pinifolia. Instead, Choi et al. [48] very few fungal species causing disease in humans. These reported the antimicrobial properties of Zostera marina infections range from superficial mucosal and dermal methanol extract and its organic solvent fractions on three infections, such as thrush, vaginal yeast infections, and human skin pathogens (Staphylococcus aureus, S. epider- diaper rash, to vascular catheters and dental implants midis and C. albicans), and Natrah et al. [47]reportedthe infections [44]. antibacterial properties of methanol extracts of E. acor- The bioactive properties of the seagrass species selected oides and other seagrass and seaweed species on different in this work are well known, and have been reported in aquaculture pathogens (Aeromonas hydrophila, Vibrio detail by several authors [45–47]. However, until now alginolyticus, V. parahaemolyticus, V. anguillarum and attention has mainly focused on the antimicrobial activity others). of seagrass extracts, which, through disk diffusion assays, In contrast, to the best of our knowledge, no papers were investigated not in their capacity as biofilm-forming have investigated the antibiofilm activity of Enhalus microorganisms but in their planktonic state. Using lethal acoroides, Halodule pinifolia and Halophila ovalis at concentrations, Umamaheshwari et al. [46]reported the non-lethal concentrations against bacterial (E. coli)and De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 10 of 17 Fig. 3 E. coli (a) and C. albicans (b) planktonic growth without (positive control) and with each seagrass extract at 100 ppm. The positive control was set up with mineral medium supplemented with glucose at 5 g/l. Stars indicate statistically significant differences (Tukey’s HSD, p ≤ 0.05) between the means of three independent replicates. (EAL = Enhalus acoroides leaf; EAR = Enhalus acoroides root; HPL = Halodule pinifolia leaf; HOL = Halophila ovalis leaf; C + = Positive control) Fig. 4 OD-based growth curves of C. albicans in absence (positive control) and in presence of each seagrass extract at 10 and 100 ppm. Maximum specific growth rate (μ )and the goodnessof fit (R ) obtained by the Gompertz model. Data represent the mean ± SDs of three independent measurements. Means reported showed no statistically significant differences between the positive control and treated samples (Tukey’sHSD, p ≥ 0.05). (EAL = Enhalus acoroides leaf; EAR = Enhalus acoroides root; HPL = Halodule pinifolia leaf; HOL = Halophila ovalis leaf; C + = Positive control) De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 11 of 17 Fig. 5 OD-based growth curves of E. coli in absence (positive control) and in presence of each seagrass extract at 10 and 100 ppm. Maximum specific growth rate (μ )and the goodnessof fit (R ) obtained by the Gompertz model. Data represent the mean ± SDs of three independent measurements. Means reported showed no statistically significant differences between the positive control and treated samples (Tukey’sHSD, p ≥ 0.05). (EAL = Enhalus acoroides leaf; EAR = Enhalus acoroides root; HPL = Halodule pinifolia leaf; HOL = Halophila ovalis leaf; C + = Positive control) fungal (C. albicans) biofilms. To this end, methanol ex- biofilm formation, microtiter based assays were per- tracts, obtained from different organs of three seagrass formed. The results revealed excellent anti-adhesion ac- species (namely, Enhalus acoroides leaves and roots, tivity for E. acoroides leaf extract, reducing fungal Halophila ovalis leaves and Halodule pinifolia leaves) coverage up to 74% and bacterial coverage up to 61% at were screened for their ability to modulate biofilm gen- 0.01 and 10 mg/l, respectively. Therefore, 0.01 mg/l and esis without killing cells. Methanol was used as the 10 mg/l E. acoroides leaf extract were chosen as the extraction solvent, having been previously reported as best non-biocidal concentrations for C. albicans and E. the most effective solvent to obtain high concentra- coli respectively, and were used in the subsequent stud- tions of bioactive compounds with antibacterial activity ies. These concentrations significantly decreased the from seagrasses, compared to other extraction solvents number of adhered cells on a hydrophobic surface, [45, 49, 50]. more so than on the hydrophilic one. Previous studies Before evaluating the antibiofilm activity, the extracts, had highlighted the preference for hydrophobic sur- at concentrations of 100 mg/l, were first proved to not faces, these reporting a decreased adhesion on the act as a carbon and energy source nor to affect the hydrophobic surface compared to the hydrophilic [51, cellular growth of C. albicans and E. coli.Therefore, 52]. This is probably due to the hydrophobic nature of concentrations ≤100 mg/l plant extract were used in the aerial surfaces of plants [53]. the subsequent studies. In the present study the anti-adhesion activity of the With the aim of investigating the effects of seagrass seagrass extracts was dose-dependent, but the highest extracts on cell adhesion to surfaces, the first step of concentrations did not correspond to those with the De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 12 of 17 Fig. 6 Microplate-based biofilm assay. Percentage reduction of the number of adhered cells of E. coli and C. albicans on hydrophilic and hydrophobic surface in presence of non-lethal concentrations of seagrass extracts. According to post hoc analysis (Tukey’s HSD, p ≤ 0.05), stars indicate statistically significant differences between the means of three independent replicates. In addition, the mean ± SDs of the percentage reduction of the number of adhered cells with seagrass extracts at non-lethal concentrations on hydrophilic and hydrophobic surface are reported in the table. The higher anti-adhesion effect for each microorganism was highlighted. (EAL = Enhalus acoroides leaf; EAR = Enhalus acoroides root; HPL = Halodule pinifolia leaf; HOL = Halophila ovalis leaf) best performance. Indeed, several studies have reported literature, is defined as hormesis, an adaptive behavior a weak activity of the compounds at low and high of microorganisms to provide resistance to environ- concentrations, and excellent activity at intermediate mental stress and improve the allocation of resources concentrations [54]. Such a response, widely known in to ensure cell stability [19, 55]. De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 13 of 17 Fig. 7 CDC biofilm growth on polycarbonate coupons (a) and biofilm dispersion rate (b)of C. albicans in absence (positive control) and in presence (treated) of 0.01 ppm of Enhalus acoroides leaf extract. Stars indicate statistically significant differences (Tukey’s HSD, p ≤ 0.05) between the means of three independent replicates. (C + = Positive control; EAL = Enhalus acoroides leaf) To further explore the effect of the most promising the number of dispersed cells in the treated biofilm, seagrass extract on biofilm development and detachment, compared with the untreated (70 ± 6.83%), suggesting CDC reactors were employed to reproduce biofilm at the a further mechanism of action for the seagrass extract solid/liquid interface, while for the assessment of the anti- as biofilm dispersing agent. In fact, the phase of bio- biofilm effect in the adhesion phase microplate-based film dispersion could be an interesting target for the biofilm assays are the most suitable [41, 56, 57]. In this development of new antibiofilm strategies, forcing the study, a significant reduction in fungal coverage (up to planktonic state and reestablishing the efficacy of 26.77 ± 9.01%) after 24 h (static adhesion phase) was traditional antimicrobial agents [4, 58]. Literature with observed in presence of 0.01 mg/l E. acoroides leaf information related to C. albicans biofilm dispersion is extract. This result confirms the anti-adhesion activity scarce. Farnesol and cis-2-decenoic acid showed observed in microtiter assays. Coupons collected after dispersion-promotion of microcolonies of C. albicans 48 and 72 h showed no significant differences between biofilm [58, 59]. In addition, Villa et al. [60]reported that treated and control samples. non-lethal concentrations of Muscari comosum ethanol In order to assess the possibility of 0.01 mg/l E. acoroides bulb extract can modulate yeast adhesion and subsequent leaf extract to promote C. albicans biofilm-detachment biofilm development on abiotic surfaces, and such con- from the surface of coupons, a biofilm dispersion assay centrations could provide an extracellular signal respon- was performed. Results showed a significant increase in sible for biofilm dispersion. Fig. 8 Biofilm growth at the solid/air interface. E. coli biofilm grown on polycarbonate membrane under three experimental conditions: i) treatment 1: growth in contact with 1 ml of LB with 10 ppm of E. acoroides leaf extract; ii) treatment 2: overnight culture grown with 10 ppm of E. acoroides leaf extract and then growth in contact with 1 ml of LB; iii) treatment 3: overnight culture grown with 10 ppm of E. acoroides leaf extract and growth in contact with 1 ml of LB with 10 ppm of E. acoroides leaf extract. In the positive control, microorganisms grew in 1 ml LB inside a basolateral well without the extract. Data obtained were divided by the area of the membrane, and means were reported. The experiment was repeated three times. (T1 = treatment 1; T2 = treatment 2; T3 = treatment 3; C + = Positive control) De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 14 of 17 Fig. 9 Relative luminescence emitted by Vibrio harveyi in absence (positive control) and in presence of 10 ppm of E. acoroides leaf extract for 24 h. The relative luminescence has been calculated by normalizing luminescence by the number of adhered cells. Stars indicate statistically significant differences (Tukey’sHSD,p ≤ 0.05) between the means of three independent replicates. (C + = Positive control; EAL = Enhalus acoroides leaf) For E. coli, the CDC reactor was not suitable to evalu- phenolic compound occurring in the seagrass Zostera ate the possible effects of the extracts on the biofilm marina. In fact, it has been hypothesized that the stages. Also other authors have reported the poor biofilm accumulation of AI-2 above a threshold level leads to formation exhibited by E. coli K-12 strain under hydro- reduced biofilm formation due to the induction of a dynamic conditions [61–63]. The effect of 10 mg/l of E. hypermotile phenotype that is unable to adhere to the acoroides leaf extract on E. coli biofilm formation was then surface [64]. Huber et al. [65] demonstrated that some evaluated using a membrane-supporting biofilm reactor, polyphenolic compounds containing a gallic acid residue which allowed the formation of a biofilm at the solid/air commonly produced by some plant species inhibited interface. This technique forced the cells to attach to a sur- intercellular communication in bacteria. Truchado et al. face, a feature that allowed direct investigation of the effect [66] reported the ability of some phytochemical com- of the selected extract on the development of the biofilm, pounds (cinnamaldehyde, ellagic acid, resveratrol, rutin whilst bypassing the effect on the adhesion phase. and pomegranate extract) to interfere with the quorum No significant reduction in the number of viable cells sensing system of Yersinia enterocolitica and Erwinia during biofilm formation on the membrane treated with cartovora. the extract, compared to the untreated, after 18 h in all It has been well known that the antibiofilm activity of the experimental conditions was observed. Treatment 3 plant extracts is closely linked with the content of sec- showed a growth rate slowdown in the interval 6–8h,in ondary metabolites, such as phenols and/or flavonoids, which E. coli cells were in contact with the extract dur- which represent the total amount of phenolic com- ing both overnight growth and biofilm formation (reduc- pounds in a plant extract [13]. The phenolic compound tion of cellular growth, compared to the control, up to content is also deeply associated with the antioxidant 48.64 ± 4.02%). Interestingly, treatment 3 showed a bi- activity of plant extracts [67]. Therefore, we determined phasic growth curve compared with the growth curves the total phenolic acid (TPC) and flavonoid (TFC) content of the other treatments, a trend that could be explained and the antioxidant activity (ORAC) of methanolic ex- by the bioluminescence produced by V. harvey. As sig- tracts in order to highlight features of the most promising naling molecules play an important role in biofilm devel- antibiofilm extract, the E. acaroides leaf extract. Results opment and detachment, the effects of 10 mg/l of E. show that E. acaroides leaf extract presents the lower TPC acoroides leaf extract were investigated using V. harveyi, and TFC values compared to other seagrasse extracts. suggesting other possible antibiofilm mechanisms of ac- Although the low content of phenolic compounds, the E. tion of compounds in the chosen seagrass extract. The acaroides leaf extract displays a higher ORAC value com- results revealed that at time 8 h, the samples treated pared to the root extract. This indicates the abundance of with the leaf extract showed a significant increase in the other, non-phenolic compounds with antioxidant capacity relative luminescence emitted, compared to the control in the leaves of E. acoroides. Cattò et al. [39] suggested the (25.75 ± 7.49). Villa et al. [64] reported an increase of importance of antioxidant compounds in hindering autoinducer-2 (AI-2) activity and a reduction in biofilm biofilm formation. The researcher discovered that the formation in E. coli cells treated with zosteric acid, a mechanism of action behind the antibiofilm performance De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 15 of 17 of zosteric acid, a secondary metabolite of the seagrass Acknowledgments We would like to thank Dr. Vy Xuan Nguyen for the identification and Zostera marina, is related to the antioxidant activity of the collection of the seagrass samples in Vietnam used in this work. molecule, and its interaction with the WrbA protein re- sponsible maintaining cellular homeostasis and defense Funding This work was supported by the German-Italian bilateral project “Bioactive against oxidative stress. secondary compounds from halophyte species inhibit biofilm formation of To gain more insight into possible antibiofilm com- plant-pathogenic microorganisms on plant surfaces” (SAB-HAL), MIUR-DAAD pounds in the seagrass extracts, individual substances in Joint Mobility Program, ID-57265315. the methanolic extract were analyzed by LC-MS. Prelim- Availability of data and materials inary analysis shows that the phytochemical profile of The datasets used and/or analyzed during the current study available from the E. acaroides leaf extract is mainly characterized by the corresponding author on reasonable request. the presence of the flavones apigenin and luteolin, three Authors’ contributions kaempferol derivates and the carboxylic acids benzoic DVL performed the biological experiments and wrote the manuscript. and azelaic acid. This unique quantitative and qualitative GY extracted and analyzed the crude extracts from the plants by MS. CC participated in the design of the study and provided technical advices and lab chemical composition confers antibiofilm properties to supports. VF conceived, designed and coordinated the study. VF contributed the E. acaroides leaf extract. substantially to the writing and revising of the manuscript. CF and PJ Some of these compounds have shown to exhibit anti- participated in the design of the study, in discussions and reviewed the manuscript. All authors read and approved the final manuscript. biofilm properties at non-lethal concentrations. Kaemp- ferol, apigenin and luteolin from red wine reduced Competing interest biofilm formation of methicillin-sensitive S. aureus sig- The authors declare that they have no competing interests. nificantly [68]. Sánchez et colleagues [69] reported that Ethics approval and consent to participate sub-lethal concentrations of plant extracts inhibit E. coli Not applicable. and S. aureus biofilms. The antibiofilm properties of the extracts were associated to the presence of flavonoids, Publisher’sNote such as kaempferol and apigenin, which modulate bac- Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. terial cell-cell communication by suppressing the activity of the autoinducer-2 [70]. However, we should keep in Author details mind that the antibiofilm effects of plant extracts could Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, Università degli Studi di Milano, via Celoria 2, 20133 Milan, Italy. Institute of be the result of interactions among different compo- Botany, Leibniz University Hannover, Herrenhäuserstr. 2, D-30419 Hannover, nents of the extract at specific concentrations, and not Germany. only due to the effects of a single, predominant com- Received: 16 December 2017 Accepted: 15 May 2018 pound [4, 71]. Conclusions References 1. Costerton JW. Introduction to biofilm. Int J Antimicrob Agents. 1999;11:217–21. In conclusion, the E. acoroides leaf extract proved to be 2. Hall-Stoodley L, Costerton WJ, Stoodley P. Bacterial biofilms: from the the most promising extract among those tested. Indeed, natural environment to infectious diseases. Nat Rev Microbiol. 2004;2:95– the selected non-lethal concentrations of E. acoroides 108. https://doi.org/10.1038/nrmicro821. 3. Rayner J, Veeh R, Flood J. Prevalence of microbial biofilms on selected fresh leaf extract were found to exert an antibiofilm effect on produce and household surfaces. Int J Food Microbiol. 2004;95:29–39. C. albicans and E. coli biofilm in the first phase of bio- 4. Villa F, Villa S, Gelain A, Cappitelli F. Sub-lethal activity of small molecules film genesis, opening up the possibility of developing from natural sources and their synthetic derivatives against biofilm forming nosocomial pathogens. Curr Top Med Chem. 2013;13(24):3184–204. preventive strategies to hinder the adhesion of micro- 5. Macià MD, Rojo-Molinero E, Oliver A. Antimicrobial susceptibility testing in bial cells to surfaces. The leaf extract also affected the biofilm-growing bacteria. Clin Microbiol Infect. 2014;20(10):981–90. https:// dispersion and maturation steps in C. albicans and E. doi.org/10.1111/1469-0691.12651. 6. Percival SL, Suleman L, Vuotto C, Donelli G. Healthcare-associated infections, coli respectively, suggesting an important role in cell medical devices and biofilms: risk. tolerance and control J Med Microbiol. signaling processes. These effects could be explained by 2015;64(Pt 4):323–34. https://doi.org/10.1099/jmm.0.000032. the presence of active compounds like kaempferol and 7. Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC. Hidden killers: human fungal infections. Sci Transl Med. 2012;4:165rv113. https://doi. apigenin at specific concentrations in the extracts of E. org/10.1126/scitranslmed.3004404. acoroides, which are known to possess biofilm inhibit- 8. Akbari F, Kjellerup BV. Elimination of bloodstream infections associated with ing properties. Furthermore, there could be a synergis- Candida albicans biofilm in intravascular catheters. Pathogens 2015;4(3):457– 69. https://doi.org/10.3390/pathogens4030457. tic action of these flavonoids with other compounds 9. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. occurring in the plant, enhancing the global antibiofilm Lancet. 2001;358:135–8. https://doi.org/10.1016/S0140-6736(01)05321-1. effect. Currently, the leaf extract is being investigated 10. Smith K, Hunter IS. Efficacy of common hospital biocides with biofilms of multi-drug resistant clinical isolates. J Med Microbiol. 2008;57:966–73. with the objective of testing fractions for identifying the https://doi.org/10.1099/jmm.0.47668-0. active compounds and to better understand the mecha- 11. Lebeaux D, Ghigo JM, Beloin C. Biofilm-related infections: bridging the gap nisms of action of this seagrass species. between clinical management and fundamental aspects of recalcitrance De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 16 of 17 toward antibiotics. Microbiol Mol Biol Rev. 2014;78(3):510–43. https://doi. 32. Dewanto V, Wu X, Adom KK, Liu RH. Thermal processing enhances the org/10.1128/MMBR.00013-14. nutritional value of tomatoes by increasing total antioxidant activity. J Agric 12. Chung PY. Plant-derived compounds as potential source of novel anti- Food Chem. 2002;50:3010–4. https://doi.org/10.1021/jf0115589. biofilm agents against Pseudomonas aeruginosa. Curr Drug Targets. 2017; 33. Huang D, Ou B, Hampsch-Woodill M, Flanagan JA, Prior RL. High- 18(4):414–20. https://doi.org/10.2174/1389450117666161019102025. throughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate 13. Choi HA, Cheong DE, Lim HD, Kim WH, Ham MH, Oh MH, Wu Y, Shin HJ, fluorescence reader in 96-well format. J Agric Food Chem. 2002;50(16): Kim GJ. Antimicrobial and anti-biofilm activities of the methanol extracts of 4437–44. https://doi.org/10.1021/jf0201529. medicinal plants against dental pathogens Streptococcus mutans and Candida albicans. J Microbiol Biotechnol. 2017;27(7):1242–8. https://doi.org/ 34. Gillespie KM, Chae JM, Ainsworth EA. Rapid measurement of total 10.4014/jmb.1701.01026. antioxidant capacity in plants. Nat Protoc. 2007;2(4):867–70. https://doi.org/ 14. da Silva DT, Herrera R, Batista BF, Heinzmann BM, Labidi J. Physicochemical 10.1038/nprot.2007.100. characterization of leaf extracts from Ocotea lancifolia and its effect against 35. Horai H, Arita M, Kanaya S, Nihei Y, Ikeda T, Suwa K, Ojima Y, wood-rot fungi. Int Biodeterior Biodegradation. 2017;117:158–70. doi.org/10. Tanaka K, Tanaka S, Aoshima K, Oda Y, Kakazu Y, Kusano M, Tohge T, 1016/j.ibiod.2016.12.007 Matsuda F, Sawada Y, Yokota Hirai M, Nakanishi H, Ikeda K, Akimoto N, 15. Teanpaisan R, Kawsud P, Pahumunto N, Puripattanavong J. Screening for Maoka T, Takahashi H, Ara T, Sakurai N, Suzuki H, Shibata D, Neumann S, antibacterial and antibiofilm activity in Thai medicinal plant extracts against Iida T, Tanaka K, Funatsu K, Matsuura F, Soga T, Taguchi R, Saito K, oral microorganisms. J Tradit Complement Med. 2017;7(2):172–7. doi.org/10. Nishioka T. MassBank: a public repository for sharing mass spectral data 1016/j.jtcme.2016.06.007 for life sciences. J Mass Spectrom. 2010;45:703–14. https://doi.org/10. 16. Abiala M, Olayiwola J, Babatunde O, Aiyelaagbe O, Akinyemi S. Evaluation of 1002/jms.1777. therapeutic potentials of plant extracts against poultry bacteria threatening 36. Sawada Y, Nakabayashi R, Yamada Y, Suzuki M, Sato M, Sakata A, Akiyama K, public health. BMC Complement Altern Med. 2016;16(1):417. Sakurai T, Matsuda F, Aoki T, Hirai MY, Saito K. RIKEN tandem mass spectral database (ReSpect) for phytochemicals: a plant-specific MS/MS-based data 17. Bisi-Johnson MA, Obi CL, Samuel BB, Eloff JN, Okoh AI. Antibacterial activity resource and database. Phytochemistry. 2012;82:38–45. https://doi.org/10. of crude extracts of some south African medicinal plants against multidrug 1016/j.phytochem.2012.07.007. resistant etiological agents of diarrhoea. BMC Complement Altern Med. 37. Zwietering MH, Jongenburger I, Rombouts FM, van’t Riet K. Modeling of the 2017;17(1):321. https://doi.org/10.1186/s12906-017-1802-4. bacterial growth curve. Appl Environ Microbiol 1990;56:1875–1881. 18. Elisha IL, Botha FS, McGaw LJ, Eloff JN. The antibacterial activity of extracts of nine plant species with good activity against Escherichia coli against five 38. Villa F, Albanese D, Giussani B, Stewart P, Daffonchio D, Cappitelli F. other bacteria and cytotoxicity of extracts. BMC Complement Altern Med. Hindering biofilm formation with zosteric acid. Biofouling. 2010;26:739–52. 2017;17(1):133. https://doi.org/10.1186/s12906-017-1645-z. https://doi.org/10.1080/08927014.2010.511197. 19. Villa F, Cappitelli F. Plant-derived bioactive compounds at sub-lethal 39. Cattò C, Dell’Orto S, Villa F, Villa S, Gelain A, Vitali A, Marzano V, Baroni S, concentrations: towards smart biocide-free antibiofilm strategies. Forlani F, Cappitelli F. Unraveling the structural and molecular basis Phytochem Rev. 2013;12:245–54. https://doi.org/10.1007/s11101-013-9286-4. responsible for the anti-biofilm activity of zosteric acid. PLoS One. 2015; 10(7):e0131519. https://doi.org/10.1371/journal.pone.0131519. 20. Flowers TJ, Colmer TD. Salinity tolerance in halophytes. New Phytol. 2008; 40. Villa F, Pitts B, Stewart PS, Giussani B, Roncoroni S, Albanese D. 179:945–63. https://doi.org/10.1111/j.1469-8137.2008.02531.x. Efficacy of zosteric acid sodium salt on the yeast biofilm model 21. Flowers TJ, Colmer TD. Plant salt tolerance: adaptations in halophytes. Ann Candida albicans. Microb Ecol. 2011;62:584–98. https://doi.org/10.1007/ Bot. 2015:115327–31. https://doi.org/10.1093/aob/mcu267. s00248-011-9876-x. 22. Joshi R, Ramanarao MV, Bedre R, Sanchez L, Pilcher W, Zandkarimi H, Baisakh N. Salt adaptation mechanisms of halophytes: improvement of salt 41. Cattò C, Grazioso G, Dell’Orto S, Gelain A, Villa S, Marzano V, Vitali A, Villa F, tolerance in crop plants. In: Pandey GK, editor. Elucidation of abiotic stress Cappitelli F, Forlani F. The response of Escherichia coli biofilm to salicylic acid. signaling in plants. New York, NY: Springer; 2015. p. 243–79. https://doi.org/ Biofouling. 2017;33:235–51. https://doi.org/10.1080/08927014.2017.1286649. 10.1007/978-1-4939-2540-7_9. 42. Garuglieri E, Meroni E, Cattò C, Villa F, Cappitelli F, Erba D. Effects of sub- lethal concentrations of silver nanoparticles on a simulated intestinal 23. Selmar D. Potential of salt and drought stress to increase pharmaceutical prokaryotic-eukaryotic interface. Front Microbiol. 2018;8:2698. https://doi. significant secondary compounds in plants. Landbauforschung. 2008;58: org/10.3389/fmicb.2017.02698. 139–44. 43. Sharma G, Sharma S, Sharma P, Chandola D, Dang S, Gupta S, Gabrani R. 24. Ksouri R, Ksouri WM, Jallali I, Debez A, Magné C, Hiroko I, Abdelly C. Escherichia coli biofilm: development and therapeutic strategies. J Appl Medicinal halophytes: potent source of health promoting biomolecules Microbiol. 2016;121(2):309–19. https://doi.org/10.1111/jam.13078. Epub with medical, nutraceutical and food applications. Crit Rev Biotechnol. 2012; 2016 Mar 11 32:289–326. https://doi.org/10.3109/07388551.2011.630647. 25. Boestfleisch C, Wagenseil NB, Buhmann AK, Seal CE, Wade EM, Muscolo A, 44. Nobile CJ, Johnson AD. Candida albicans biofilms and human disease. Annu Papenbrock J. Manipulating the antioxidant capacity of halophytes to Rev Microbiol. 2015;69:71–92. https://doi.org/10.1146/annurev-micro- increase their cultural and economic value through saline cultivation. AoB 091014-104330. Plants. 2014;6:plu046. https://doi.org/10.1093/aobpla/plu046. 45. Kumar SC, Sarada DVL, Gideon TP, Rengasamy R. Antibacterial activity of 26. Hua KF, Hsu HY, Su YC, Lin IF, Yang SS, Chen YM. Study on the three south Indian seagrasses, Cymodocea serrulata, Halophila ovalis and antinflammatory activity of methanol extract from seagrass Zostera japonica. Zostera capensis. World J Microbiol Biotechnol. 2008;24(9):1989–92. https:// J Agric Food Chem. 2006;54:306–11. https://doi.org/10.1021/jf0509658. doi.org/10.1007/s11274-008-9695-5. 46. Umamaheshwari R, Thirumaran G, Anantharaman P. Potential antibacterial 27. Gokce G, Haznedaroglu MZ. Evaluation of antidiabetic, antioxidant and activities from vellar estuary; south east coast of India. Adv Biomed Res. vasoprotective effects of Posidonia oceanica extract. J Ethnopharmacol. 2009;3:140–3. 2008;115:122–30. 47. Natrah FMI, Harah ZM, Japar Sidik NI, Syahidah A. Antibacterial 28. Kannan RRR, Arumugam R, Anantharaman P. Chemical composition and activities of selected seaweed and seagrass from port Dickson coastal antibacterial activity of Indian seagrasses against urinary tract pathogens. Food water against different aquaculture pathogens. Sains Malays. 2015; Chem. 2012;135:2470–3. https://doi.org/10.1016/j.foodchem.2012.07.070. 29. Nguyen XV, Höfler S, Glasenapp Y, Thangaradjou T, Lucas C, Papenbrock J. 44(9):1269–73. New insights into the DNA barcoding of seagrasses. Syst Biodivers. 2015;13: 48. Choi HG, Lee JH, Park HH, Sayegh FAQ. Antioxidant and antimicrobial 496–508. https://doi.org/10.1080/14772000.2015.1046408. activity of Zostera marina L. extract. Algae. 2009;24(3):179–84. https://doi. org/10.4490/algae.2009.24.3.179. 30. Lucas C, Thangaradjou T, Papenbrock J. Development of a DNA barcoding 49. Lustigman B, Brown C. Antibiotic production by marine algae isolated from system for seagrasses: successful but not simple. PLoS One. 2012;7:e29987. the New York/New Jersey coast. Bull Environ Contam Toxicol. 1991;46:329– https://doi.org/10.1371/journal.pone.0029987. 35. https://doi.org/10.1007/BF01688928. 31. Dudonné S, Vitrac X, Coutière P, Woillez M, Mérillon JM. Comparative study 50. Sastry VMVS, Rao GRK. Antibacterial substances from marine algae: of antioxidant properties and total phenolic content of 30 plant extracts of successive extraction using benzene, chloroform and methanol. Bot Mar. industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J Agric 1994;37:357–60. https://doi.org/10.1515/botm.1994.37.4.357. Food Chem. 2009;57:1768–74. https://doi.org/10.1021/jf803011r. De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 17 of 17 51. Doss RP, Potter SW, Chastagner GA, Christian JK. Adhesion of nongerminated 70. Vikram A, Jayaprakasha GK, Jesudhasan PR, Pillai SD, Patil BS. Suppression of Botrytis cinerea conidia to several substrata. Appl Environ Microbiol. 1993;59: bacterial cell-cell signalling, biofilm formation and type III secretion system 1786–91. by citrus flavonoids. J Appl Microbiol. 2010;109(2):515–27. https://doi.org/10. 52. Amiri A, Cholodowski D, Bompeix G. Adhesion and germination of 1111/j.1365-2672.2010.04677.x. waterborne and airborne conidia of Penicillium expansum to apple and inert 71. Bazargani MM, Rohloff J. Antibiofilm activity of essential oils and plant surfaces. Physiol Mol Plant Pathol. 2005;67:40–8. https://doi.org/10.1016/j. extracts against Staphylococcus aureus and Escherichia coli biofilms. Food pmpp.2005.07.003. Control. 2016;61:156–64. https://doi.org/10.1016/j.foodcont.2015.09.036. 53. Koch K, Bhushan B, Barthlott W. Multifunctional surface structures of plants: an inspiration of biomimetics. Prog Mater Sci. 2009;54:137–78. https://doi. org/10.1016/j.pmatsci.2008.07.003. 54. Rickard AH, Palmer RJ, Blehert DS, Campagna SR, Semmelhack MF, Egland PG, Bassler BL, Kolenbrander PE. Autoinducer 2: a concentration-dependent signal for mutualistic bacterial biofilm growth. Mol Microbiol. 2006;60:1446– 56. https://doi.org/10.1111/j.1365-2958.2006.05202.x. 55. Calabrese EJ, Baldwin LA. Hormesis: the dose-response revolution. Annu Rev Pharmacol Toxicol. 2003;43:175–97. https://doi.org/10.1146/annurev. pharmtox.43.100901.140223. 56. Williams DL, Woodbury KL, Haymond BS, Parker AE, Bloebaum RD. A modified CDC biofilm reactor to produce mature biofilms on the surface of PEEK membranes for an in vivo animal model application. Curr Microbiol. 2011;62:1657–63. https://doi.org/10.1007/s00284-011-9908-2. 57. Coffey BM, Anderson GG. Biofilm formation in the 96-well microtiter plate. Methods Mol Biol. 2014;1149:631–41. https://doi.org/10.1007/978-1-4939- 0473-0_48. 58. Davies DG, Marques CN. A fatty acid messenger is responsible for inducing dispersion in microbial biofilms. J Bacteriol. 2009;191:1393–403. https://doi. org/10.1128/JB.01214-08. 59. Uppuluri P, Chaturvedi AK, Srinivasan A, Banerjee M, Ramasubramaniam AK, Köhler JR, Kadosh D, Lopez-Ribot JL. Dispersion as an important step in the Candida albicans biofilm developmental cycle. PLoS Pathog. 2010;6: e1000828. https://doi.org/10.1371/journal.ppat.1000828. 60. Villa F, Borgonovo G, Cappitelli F, Giussani B, Bassoli A. Sublethal concentrations of Muscari comosum bulb extract suppress adhesion and induce detachment of sessile yeast cells. Biofouling. 2012;28:1107–17. https://doi.org/10.1080/08927014.2012.734811. 61. Ghigo JM. Natural conjugative plasmids induce bacterial biofilm development. Nature. 2001;412:442–5. https://doi.org/10.1038/35086581. 62. Reisner A, Haagensen JA, Schembri MA, Zechner EL, Mølin S. Development and maturation of Escherichia coli K-12 biofilms. Mol Microbiol. 2003;48:933– 46. https://doi.org/10.1046/j.1365-2958.2003.03490.x. 63. Reisner A, Krogfelt KA, Klein BM, Zechner EL, Mølin S. 2006. In vitro biofilm formation of commensal and pathogenic Escherichia coli strains: impact of environmental and genetic factors. J Bacteriol. 2006;188:3572–81. https://doi. org/10.1128/JB.188.10.3572-3581.2006. 64. Villa F, Remelli W, Forlani F, Vitali A, Cappitelli F. Altered expression level of Escherichia coli proteins in response to treatment with the antifouling agent zosteric acid sodium salt. Environ Microbiol. 2012;14:1753–61. 65. Huber AB, Kolodkin AL, Ginty DD, Cloutier JF. Signaling at the growth cone: ligand-receptor complexes and the control of axon growth and guidance. Annu Rev Neurosci. 2003;26:509–63. https://doi.org/10.1146/annurev.neuro. 26.010302.081139. 66. Truchado P, Lopez-Galvez F, Gil MI, Tomas-Barberan FA, Allende A. Quorum sensing inhibitory and antimicrobial activities of honeys and the relationship with individual phenolics. Food Chem. 2009;115:1337–44. https://doi.org/10.1016/j.foodchem.2009.01.065. 67. Reis Giada ML. Food phenolic compounds: main classes, sources and their antioxidant power, oxidative stress and chronic degenerative diseases Jose Antonio Morales-Gonzalez, IntechOpen. 2013. https://doi.org/10.5772/51687. Available from: https://www.intechopen.com/books/oxidative-stress-and- chronic-degenerative-diseases-a-role-for-antioxidants/food-phenolic- compounds-main-classes-sources-and-their-antioxidant-power. 68. Cho HS, Lee JH, Cho MH, Lee J. Red wines and flavonoids diminish Staphylococcus aureus virulence with anti-biofilm and anti-hemolytic activities. Biofouling. 2015;31(1):1–11. https://doi.org/10.1080/08927014. 2014.991319. 69. Sánchez E, Rivas Morales C, Castillo S, Leos-Rivas C, García-Becerra L, Mizael Ortiz Martínez D. Antibacterial and antibiofilm activity of methanolic plant extracts against nosocomial microorganisms. J Evid Based Complementary Altern Med. 2016, Article ID 1572697;2016:8. https://doi.org/10.1155/2016/ http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Complementary and Alternative Medicine Springer Journals

Hindering the formation and promoting the dispersion of medical biofilms: non-lethal effects of seagrass extracts

Free
17 pages

Loading next page...
 
/lp/springer_journal/hindering-the-formation-and-promoting-the-dispersion-of-medical-y1kvFfWbRS
Publisher
Springer Journals
Copyright
Copyright © 2018 by The Author(s).
Subject
Medicine & Public Health; Complementary & Alternative Medicine; Internal Medicine; Chiropractic Medicine
eISSN
1472-6882
D.O.I.
10.1186/s12906-018-2232-7
Publisher site
See Article on Publisher Site

Abstract

Background: Biofilms have great significance in healthcare-associated infections owing to their inherent tolerance and resistance to antimicrobial therapies. New approaches to prevent and treat unwanted biofilms are urgently required. To this end, three seagrass species (Enhalus acoroides, Halophila ovalis and Halodule pinifolia) collected in Vietnam and in India were investigated for their effects in mediating non-lethal interactions on sessile bacterial (Escherichia coli) and fungal (Candida albicans) cultures. The present study was focused on anti-biofilm activities of seagrass extracts, without killing cells. Methods: Methanolic extracts were characterized, and major compounds were identified by MS/MS analysis. The antibiofilm properties of the seagrass extracts were tested at sub-lethal concentrations by using microtiter plate adhesion assay. The performance of the most promising extract was further investigated in elegant bioreactors to reproduce mature biofilms both at the solid/liquid and the solid/air interfaces. Dispersion and bioluminescent assays were carried out to decipher the mode of action of the bioactive extract. Results: It was shown that up to 100 ppm of crude extracts did not adversely affect microbial growth, nor do they act as a carbon and energy source for the selected microorganisms. Seagrass extracts appear to be more effective in deterring microbial adhesion on hydrophobic surfaces than on hydrophilic. The results revealed that non-lethal concentrations of E. acoroides leaf extract: i) reduce bacterial and fungal coverage by 60.9 and 73.9%, respectively; ii) affect bacterial biofilm maturation and promote dispersion, up to 70%, in fungal biofilm; iii) increase luminescence in Vibrio harveyi by 25.8%. The characterization of methanolic extracts showed the unique profile of the E. acoroides leaf extract. Conclusions: E. acoroides leaf extract proved to be the most promising extract among those tested. Indeed, the selected non-lethal concentrations of E. acoroides leaf extract were found to exert an antibiofilm effect on C. albicans and E. coli biofilm in the first phase of biofilm genesis, opening up the possibility of developing preventive strategies to hinder the adhesion of microbial cells to surfaces. The leaf extract also affected the dispersion and maturation steps in C. albicans and E. coli respectively, suggesting an important role in cell signaling processes. Keywords: Seagrass extracts, Non-lethal concentrations, Antibiofilm activity, Escherichia coli, Candida albicans * Correspondence: federica.villa@unimi.it Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, Università degli Studi di Milano, via Celoria 2, 20133 Milan, Italy Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 2 of 17 Background extracts, including antibacterial, antifungal, antialgal, anti- The ability of microorganisms to colonize surfaces and oxidant, anti-inflammatory, insecticidal, antimalarial and develop into highly organized communities enclosed in a vasoprotective properties, have been reported [26–28]. self-produced polymeric matrix is the predominant Thus, the well described properties of seagrasses ex- growth modality in both nature and artificial systems. tracts offer a promising framework for investigating novel Such lifestyle is called biofilm and it is characterized by antibiofilm activities at non-lethal concentrations. alterations in microbial phenotypes with respect to growth The present study explores, for the first time, the effect rates and gene transcriptions [1–3]. of extracts from different seagrasses (namely, leaves and Biofilms have great significance for public health, roots from Enhalus acoroides Rich. ex Steud., Hydrochari- representing 65–80% of microbial diseases currently taceae, leaves of Halophila ovalis (R.Br.) Hook.f., Hydro- treated by physicians in the developed world [4, 5]. The charitaceae, and leaves of Halodule pinifolia (Miki) presence of indwelling medical devices further increases Hartog, Cymodaceaceae) in mediating non-lethal interac- the risk for biofilm formation and subsequent infection tions on sessile Candida albicans and Escherichia coli [6]. The bacterium Escherichia coli and the polymorphic cultures, selected as model systems for fungal and bacter- fungus Candida albicans are among the most frequent ial biofilm infections, respectively. The work focuses on cause of bloodstream infections, and the predominant investigating the antibiofilm performance of seagrass ex- microorganisms isolated from infected medical devices tracts at sub-inhibitory concentrations, studying how they [7, 8]. These biofilms, as any other biofilm, exhibit affect biofilm functional traits (such as adhesion, biofilm dramatically decreased susceptibility to antimicrobial maturation, dispersal and quorum sensing), and induce agents and resistant to the host immune clearance, which cellular responses other than those associated with anti- increases the difficulties for the clinical treatment of infec- microbial activities. tions [9–11]. Furthermore, the antimicrobial arena is ex- periencing a shortage of lead compounds, and growing Methods negative consumer perception against synthetic products Plant material and extraction has led to the search for more natural solutions [12]. Three species of seagrasses (leaves and roots from In this context, it has been reported that plant-derived Enhalus acoroides Rich. ex Steud., Hydrocharitaceae, extracts exhibit good antibiofilm properties against a leaves of Halophila ovalis (R.Br.) Hook.f., Hydrocharita- range of microorganisms [13–15]. However, in the past, ceae, and leaves of Halodule pinifolia (Miki) Hartog, these extracts were mainly screened by focusing on their Cymodaceaceae) were collected in Vietnam and India lethal effects [16–18] disregarding their activity at and air-dried in a dark place (Table 1). Enhalus acor- non-lethal concentrations. At these concentrations, oides and Halophila ovalis were collected and identified plant-derived extracts may reveal elegant mechanisms by Xuan-Vy Nguyen, Department of Marine Botany, to sabotage the sessile lifestyle, manipulating the ex- Institute of Oceanography, Vietnam Academy of Science pression of stage-specific biofilm phenotypes [19]. For and Technology, Nha Trang City, Vietnam, based on mor- instance, by affecting the cellular ability to attach to phological characters and controlled by ITS molecular surfaces and by mystifying intercellular signals, the marker analysis [29]. Specimens of Enhalus acoroides are biofilm cascade might be hampered. Thus, non-lethal stored in the herbarium of the Institute of Botany, concentrations of plant-derived extracts can inspire in- Hannover, Germany (Specimen number: EA20130301). novative, eco-friendly and safe strategies aim at treating Halodule pinifolia was collected by Jutta Papenbrock and deleterious biofilms. Interfering with specific key steps further identified by Thirunavakkarasu Thangaradjou, that orchestrate biofilm genesis might offer new ways Centre of Advanced Study in Marine Biology, Annamalai to disarm microorganisms without killing them, side- University, Parangipettai, Tamilnadu, India, based on mor- stepping drug resistance [4]. phological characters and controlled by ITS molecular Seagrasses, which belong to the halophytes, represent a marker analysis [30]. Specimens are stored in the functional group of underwater marine flowering plants herbarium of the Annamalai University, Parangipettai, that have developed several strategies to survive and re- Tamilnadu, India. produce in environments where the salt concentration is The plants were separated into different organs (leaves around 200 mM NaCl or more [20]. As these plants grow and roots), and samples were cooled with liquid nitrogen in very high saline conditions, it is predicted that they and ground to a fine powder using a bead mill (Retsch), could possess rare and new activities not reported for their three times for 10 s at a frequency of 30/s. The samples terrestrial relatives [21, 22]. Indeed, metabolomic studies were stored at − 80 °C prior to analysis. Crude extracts have shown that increased salinity leads to changes in were obtained using 80% methanol (MeOH) as solvent. conserved and divergent metabolic responses in halo- Around 50 mg of powdered seagrass material was weighed phytes [23–25]. Moreover, interesting activities of seagrass in a reaction tube and extracted with 800 μl80% MeOH De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 3 of 17 Table 1 Seagrass species and information about collection sites Species Plant organ Collection site GPS Collection date Enhalus acoroides Leaf Nha Trang Bay, Vietnam 109.209208°E 19.04.2011 12.158073°N Enhalus acoroides Root Nha Trang Bay, Vietnam 109.209208°E 19.04.2011 12.158073°N Halophila ovalis Leaf Nha Trang Bay, Vietnam 109.209208°E 19.04.2011 12.158073°N Halodule pinifolia Leaf Chilika Lagoon, India 85.418015°E 16.02.2010 19.775105°N for 10 min with regular shaking. Then the extract was Quantification of total phenolic contents (TPC) centrifuged for 5 min at 18000 x g and the supernatant To measure the total phenolic acid content, a modified transferred into a new reaction tube. These steps were re- protocol after Dewanto et al. [32] was used with the peated three times with 400 μl 80% MeOH each. The su- same extracts described above. 96-well microtiter plate pernatants were collected in the same reaction tube and were filled with 100 μlH O each. From each sample, stored at − 20 °C. Phosphate buffered saline (PBS, 0.01 M 10 μl were added; seagrass extracts were diluted 1:2. A phosphate buffer, 0.0027 M potassium chloride 0.137 M, gallic acid calibration curve with the following concen- sodium chloride, Fisher Scientific) was used to obtain sev- tration was used: 0, 5, 10, 25, 50, 75, 100, 125 and eral concentrations of each crude extract: 100, 10, 1, 0.1, 250 μg/ml. Next, 100 μlNa CO 7% were added and the 2 3 0.01 and 0.001 mg/l. plate was incubated for 100 min in the dark. The ab- sorption was measured at 765 nm in a microplate reader. With the slope of the gallic acid calibration Microbial strains and growth media curve, the concentration of phenolic acids was calculated The microbial strains Candida albicans SC5314 (ATCC in mg gallic acid equivalent. MYA-2876) and Escherichia coli K-12 wild-type strain (ATCC 25404) were selected as model systems for fun- Determination if the oxygen radical absorbance capacity gal and bacterial biofilms respectively. C. albicans and (ORAC) E. coli strains were stored at − 80 °C in suspensions The analysis of the oxygen radical absorbance capacity containing 50% glycerol and 2% peptone, and were rou- (ORAC) was conducted according to a protocol based tinely grown in amino acid-free yeast nitrogen base (YNB, on Huang et al. (2002) [33] and Gillespie et al. [34] Sigma-Aldrich) supplemented with 0.5% glucose (YNBG, with the same extracts. A black 96-well microtiter was Conda) and Luria-Bertani broth (LB, Sigma-Aldrich), used and the wells were filled with 120 μl fluorescein respectively, for 16 h at 30 °C. (112 nM) in phosphate buffer (75 mM, pH 7.4). Of each sample and the standard curve, 20 μl were added Quantification of total flavonoid contents (TFC) in each well. The standard curve of 6-hydroxy-2, The total flavonoid content of the seagrass extracts was 5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) measured in 96-well plate according to a modified was prepared in phosphate buffer with the following protocol from Dudonné et al. [31]. The wells were filled concentrations: 6.25, 12.5, 25 and 50 μM. Seagrass with 150 μlH O each. Dilutions of the methanolic sea- extracts were diluted 1:200 with methanol 80%. The grass extracts (1:2) were prepared and 25 μl of sample microtiter plate was incubated for 15 min at 37 °C. The were filled in one well, with four replicates. A calibration fluorescence was then measured at 485/520 nm as time curve with catechin hydrate with the following concen- point zero. Next, 80 μlof 2,2′-azobis(2-amidino-propane) trations was prepared in 80% MeOH: 0, 10, 25, 50, 100, dihydrochloride (62 mM) were added and the fluores- 125, 250 and 400 μg/ml. The calibration curve was cence was measured every minute for 80 min. The ORAC placed on the plate in triplicate. In the next step, 10 μl value was calculated as the difference between time point NaNO 3.75% were added into each well and incubated zero and 80 min and quantified with the Trolox standard for 6 min. Afterwards, 15 μl of AlCl 10% were added curve. and incubated for 10 min. In the last step, 50 μlof NaOH 1 M were added and the absorption was mea- LC-MS analysis sured at 510 nm in a microplate reader (Biotek, Winoo- LC-MS analysis was performed on a Shimadzu HPLC ski, USA). The slope of the calibration curve was used to system (controller CBM-20A, two pumps LC-20 AD, a calculate the total flavonoid content in mg catechin column oven CTO-20 AC and a photo diode array equivalent. detector SPD-M20A; Shimadzu, Darmstadt, Germany) De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 4 of 17 coupled to a Triple Tof 4600 mass spectrometer (AB 60 min for 30 h in wells inoculated with 45 μl (3% vol/vol) Sciex, Canby, USA). The separation of extracted com- of an overnight culture (approximately 10 cells/ml). The pounds was realised on a Knauer Vertex Plus column negative control was represented by PBS supplemented (250 × 4 mm, 5 μm particle size, packing material with 45 μl (3% vol/vol) of the overnight culture. The ProntoSIL 120–5 C18-H) with precolumn (Knauer, polynomial Gompertz model [37] was used to fit the Berlin, Germany). The column oven temperature was growth curves to calculate the maximum specific growth set to 30 °C and 25 μl of undiluted methanolic seagrass rate (A /min), using GraphPad Prism software (version extract prepared as described above was injected. The 5.0, San Diego, CA, USA). Five biological replicates of solvent flow rate was 0.8 ml/min. In this time, a gradi- each treatment were performed. ent was run from 10 to 90% B from minute 0 to 35, 2min of90% B, switch to 10%Bin1min andsubse- Microplate-based biofilm assay quent equilibration at 10% B for 2 min. Solvent A The antibiofilm activity of seagrass extracts was assessed (water) and B (methanol) were both supplemented with quantitatively as previously reported by Villa et al. [38]. 2 mM ammonium acetate and 0.01% acetic acid. Mass Briefly, 200 μl of PBS containing 10 cells/ml supple- spectra were monitored between 100 and 800 Da in mented with 0 (positive control), 100, 10, 1, 0.1, 0.01, negative ionisation mode. In addition, MS/MS spectra and 0.001 mg/l of each crude extract were placed in were generated with a collision energy of − 30 eV and hydrophobic and hydrophilic 96-well polystyrene-based measured between 50 and 800 Da. Spectra for the most microtiter plates (Thermo Fisher Scientific). After an prominent peaks were compared to database entries in incubation time of 24 h at 20 °C, C. albicans and E. coli MassBank [35]and ReSpect [36] for identification. planktonic cells were removed and adhered cells were stained using 0.1 mg/ml of Fluorescent Brightener 28 vital Planktonic growth in the presence of seagrass extracts as dye (Sigma-Aldrich) or 4′, 6-diamidino-2-phenylindole the sole source of carbon and energy (DAPI, Sigma-Aldrich) in PBS, respectively. After 20 min The ability of C. albicans and E. coli planktonic cells to staining in the dark at room temperature the microtiter grow in the presence of each extract as the sole carbon plates were washed twice with 200 μl PBS and the fluores- and energy source was tested using YNB and M9 cence intensity due to adhered cells was measured using a (Sigma-Aldrich) mineral medium, respectively, supple- fluorescence microplate reader (TECAN, Manneford, mented with the highest working extract concentration: Switzerland) at excitation wavelength of 335 nm and emis- 100 mg/l. Then a 100 μl mix of mineral medium to- sion wavelength of 433 nm. A standard curve of fluores- gether with 45 μl (3% v/v) of the overnight culture (final cence intensity versus cell number was determined and concentration 10 cells/ml) and the highest concentra- used to quantify the antibiofilm performance of the crude tion of each marine plant extract were used to fill each extracts. Percentage reduction with respect to the positive well of 96-well plates (Thermo Fisher Scientific) and in- control is calculated as (treated data –control data) × 100 cubated for 48 h at 30 °C. A medium complemented / control data. Cattò et al. [39] proposed the following with cells and glucose (5 g/l), and medium without cells, anti-adhesion ranges computing the percentage reduction were used as positive and negative controls, respectively. in comparison to the negative control: ≤20% without Microbial growth was monitored using the PowerWave anti-adhesion activity; between 20 and 30% and 30 and XS2 microplate reader (Biotek) measuring the absorb- 40% low anti-adhesion activity and with moderate ance at 600 nm (A ) every 10 min. Six biological repli- anti-adhesion activity respectively; ≥40% adhered cells cates of each treatment were performed. The obtained with excellent anti-adhesion activity. Five biological repli- data were normalized to the negative control and re- cates were performed for each condition and a percentage ported as the mean of these. reduction in comparison to the negative control was calculated as (treated data – positive control data) × 100/ Growth inhibition assay in the presence of seagrass positive control data. The experiment was repeated three extracts times. The ability of the seagrass extracts to inhibit the plank- tonic growth of the selected microorganisms was investi- Biofilm growth at the solid/liquid interface gated. For this, C. albicans and E. coli were grown YNBG The most promising plant extracts were screened for and LB broth respectively without (positive control) and their effects on biofilm development. C. albicans biofilm with the highest working concentrations (10 and 100 mg/ was grown in the CDC biofilm reactor (Biosurface l) in 96-well plates (Thermo Fisher Scientific). Growth Technologies, Bozeman, MT, USA) as previously de- curves at 30 °C were generated using Infinite® F200 PRO scribed by Villa et al. [40]. Briefly, two bioreactors hosting microplate reader (TECAN, Mannedorf, Switzerland) by 24 polycarbonate coupons (to simulate a hydrophobic measuring the optical density at 600 nm (OD ) every surface) were filled with YNBG and 1 ml of overnight 600 De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 5 of 17 planktonic culture (approximately 10 cells/ml) and, in (plate well). Biofilm formation was performed at 37 °C in one of them, 0.01 mg/l of E. acoroides leaf extract was aerobic conditions for 16 h. At different time points (0, 4, added. Bioreactors were maintained under static condi- 6, 8, 16 h) some membranes were removed, biofilm was tions (no flow) for 24 h under mild stirring at 37 °C, pro- scraped off using a sterile loop, put inside a tube contain- moting fungal adhesion to the surface of the removable ing 1 ml of PBS and then homogenized twice using a polycarbonate coupons. After that, the dynamic phase was homogenizer (IKA T10 basic Ultra-Turrax – Cole-Parmer initiated and diluted YNGB was fluxed for 48 h at flow Instrument Company). Then serial dilutions were pre- rate of 250 ml/h. Biofilm growth in the absence (positive pared and 10 μl were inoculated in petri dishes containing control) and presence of the extract was evaluated by LB with agar following the drop counting method. After quantification of the biomass. At different time steps (24, 12 h at 37 °C, E. coli colonies were counted and the bio- 48 and 72 h) some polycarbonate coupons were collected mass was quantified. This assay was assessed under three in aseptic conditions and resuspended in 3 ml of PBS experimental conditions: i) treatment 1: growth in contact each. Subsequently, serial dilutions were carried out, and with 1 ml of LB with 10 mg/l of E. acoroides leaf extract 10 μl were inoculated in petri dishes containing Tryptic for 16 h; ii) treatment 2: overnight culture grown with Soy Broth medium (TSB, Sigma-Aldrich) complemented 10 mg/l of E. acoroides leaf extract, and then growth in with agar (Merck) following the drop counting method. contact with 1 ml of LB for 16 h; iii) treatment 3: over- After 12 h at 30 °C, C. albicans colonies were counted and night culture grown with 10 mg/l of E. acoroides leaf ex- the data obtained were normalized to the coupon area, tract, and then growth in contact with 1 ml of LB with and means were reported. The same protocol was used to 10 mg/l of E. acoroides leaf extract for 16 h. In the positive obtain mature biofilm of E. coli, using LB as a medium, control, the microorganisms grew in 1 ml LB inside a and evaluating 10 mg/l of E. acoroides leaf extract. Each basolateral well for 16 h without the extract. The data ob- experiment was repeated three times. tained were divided by the area of the membrane, and the means were reported. The experiment was repeated three Biofilm dispersion assay times. Mature C. albicans biofilm was grown in the CDC reactor in the absence (positive control) and presence B2ioluminescence assay using Vibrio harveyi and of 0.01 mg/l of E. acoroides leaf extract as reported Two hundred μl of autoinducer bioassay (AB) mineral below. As previously described by Cattò et al. [41], after medium (0.3 M NaCl, 0.05 M MgSO ,0.5%casein 72 h polycarbonate coupons were collected, immersed in hydrolysate, 10 μMKH PO ,1 μM L-arginine, 50% 2 4 27 ml of PBS for one minute at room temperature, serial glycerol, 0.01 μg/ml riboflavin, 1 μg/ml thiamine. pH 7. dilutions were carried out and 10 μl were inoculated in Sigma-Aldrich) containing 10% (V/V) of a tenfold dilu- petri dishes containing TSB supplemented with agar tion of an overnight culture of Vibrio harveyi BB170 (Merck) following the drop counting method. After 12 h (ATCC BAA-1117) grown in AB medium were supple- at 30 °C, C. albicans colonies were counted and the per- mented with 10 mg/l of E. acoroides leaf extract re- centage of biofilm dispersion was calculated as (number spectively, and were placed in hydrophobic 96-well of viable cells from bulk PBS × 100) / (number of viable polystyrene-based microtiter plates (Thermo Fisher cells from bulk PBS + number of viable cells from the Scientific) with transparent bottom. The positive control coupon biofilm) and means were reported. Three bio- was an AB mineral medium supplemented with 10% (V/ logical replicates were performed for each treatment and V) tenfold dilution of the overnight culture. Absorbance six technical replicates were performed for each experi- (OD ) and luminescence were measured using a mi- 600nm ment. The experiment was performed three times. croplate reader (VICTOR™X, Perkin Elmer, USA) every 8 h for 24 h, incubating the microtiter plate at 30 °C Biofilm growth at the solid/air interface during the experiment. The data obtained were normal- E. coli biofilm was grown on a sterile polycarbonate mem- ized to the number of viable cells, divided by the area of brane (PC, Whatman Nucleopore, diameter 2.5 cm, pore the membrane, and the means reported. The experiment diameter 0.2 μm) as previously described by Garuglieri et was repeated three times. al. [42]. Briefly, 0.05 ml of an overnight culture (approxi- mately 10 cells/ml) were inoculated at the center of a sterile polycarbonate membrane and, when the inoculum Statistical analysis was completely dried, the membrane was carefully put in- To evaluate statistically significant differences among side a transwell structure (ThinCert™ Cell Culture Inserts samples, analysis of variance (ANOVA) via MATLAB with translucent PET membrane – Greiner bio-one) inlaid software (Version 7.0, The MathWorks Inc., Natick, in a 6 well culture plate (Greiner bio-one). One ml of LB USA) was applied. Tukey’s honestly significant different medium was inoculated in the basolateral compartment test (HSD) was applied for pairwise comparison to De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 6 of 17 establish the significance of the data. Statistically sig- of E. acoroides also contained two kaempferol-based flavo- nificant results were represented by P values ≤0.05. noles and luteolin and also a procyanidin and a flavanole (epicatechin). In H. ovalis three flavonoids and one phen- Results olic acid was found. H. pinifolia contained several flavo- Seagrass extracts contain phenolic compounds and show noles, either based on kaempferol or quercetin and also antioxidant capacities epicatechin. The methanolic extracts from the seagrass material contained phenolic acids as well as flavonoids (Fig. 1a-b). Seagrass extracts are not used as carbon and energy The content of phenols and flavonoids was highest in H. source by C. albicans and E. coli and do not affect their pinifolia leaf extracts with 18.0 ± 0.25 and 14.3 ± 0.25 mg/ planktonic growth g dry mass (DM), respectively. In E. acoroides, the root C. albicans and E. coli planktonic cells grown only in the material showed higher amounts of total flavonoids and presence of medium supplemented with glucose were phenols than the leaf material. For all seagrass species, the used as the positive control of the experiment (Fig. 3). content of phenolic acids was higher than the flavonoid Note that the mineral medium supplemented with the content with respect to the DM. highest concentration of tested plant extracts did not Methanolic extracts from the four seagrass species promote the growth of the selected microorganisms. were analyzed for their antioxidant capacity (Fig. 1c). The response of the planktonic growth of the selected All tested extracts had the ability to absorb oxygen rad- microorganisms in the presence of the seagrass extracts at icals. H. pinifolia showed the highest activity with 97.7 the highest concentrations (10 and 100 mg/l) is reported ± 2.7 mg Trolox equivalents (TE)/g DM. E. acoroides in Figs. 4 and 5. C. albicans and E. coli growth rates (table and H. ovalis leaf extracts showed similar antioxidant in Figs. 4 and 5) showed that there are no statistically sig- capacities with 70.2 ± 4.1 and 72.5 ± 2.9 mg TE/g DM, nificant differences between the presence and the absence respectively. The root extract from E. acoroides displayed of the extracts obtained from every plant portion at any a lower ORAC value than the extract from the leaves tested concentration. Therefore, concentrations ≤100 mg/l (45.1 ± 3.2 mg TE/g DM). plant extract were used in the subsequent studies. LC-MS analysis of secondary metabolites E. acoroides leaf extract inhibits cell adhesion on a E. acoroides, H. ovalis and H. pinifolia show different hydrophobic surface compositions of secondary metabolites (Fig. 2). The The percentage reduction of the number of adhered cells identification of individual compounds in the methanolic of E. coli and C. albicans on hydrophilic and hydrophobic extracts was done via the comparison of MS/MS spectra surface in presence of non-lethal concentrations of sea- with database entries. The three seagrass species showed grass extracts is showed in Fig. 6. The results revealed that different profiles of secondary metabolites, in this case E. acoroides and H. ovalis were the most promising ex- mainly flavonoids and phenolic acids (Table 2). In E. acor- tracts for C. albicans, with excellent anti-adhesion activity, oides leaves, three flavonoles based on kaempferol were reducing fungal coverage up to 73.89 ± 1.01% and 68.37 ± found. In addition, two flavones (apigenin and luteolin), 2.49% at 0.01 and 1 mg/l, respectively. For E. coli,10 mg/l one phenolic acid (benzoic acid) and the saturated dicar- of E. acoroides leaf extract was found to be the concentra- boxylic acid azelaic acid were identified. The root extract tion with the highest reduction in cell adhesion (reduction Fig. 1 Crude methanolic extracts were analyzed for (a) Total phenols in mg gallic acid equivalent (GAE) per g dry mass (DM), (b) Total flavonoids in mg catechin equivalent (CE) per g DM, and (c) ORAC in mg Trolox equivalents (TE) per g DM. Data represent the mean ± SDs and different superscript letters indicate statistically significant differences (Tukey’s HSD, p ≤ 0.05) between the means of three independent measurements. (EAL = Enhalus acoroides leaf; EAR = Enhalus acoroides root; HPL = Halodule pinifolia leaf; HOL = Halophila ovalis leaf) De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 7 of 17 Fig. 2 Chromatograms from E. acoroides leaf extract (a), E. acoroides root extract (b), H. ovalis leaf extract (c) and H. pinifolia leaf extract (d) from minute 0–33. The relative intensity of mass between 100 and 800 Da is shown. Numbers indicate putatively identified substances in Table 2 of bacterial coverage by 60.86 ± 8.85%). Therefore, 0.01 mg/ were in contact with the extract during both overnight l and 10 mg/l E. acoroides leaf extract were chosen as the growth and biofilm formation (reduction of cellular best non-biocidal concentrations for C. albicans and E. coli growth, compared to the control, up to 48.64 ± 4.02%). respectively, and were used in the subsequent studies. This growth curve was characterized by two exponen- tial phases separated distinctly by an intermediate E. acoroides leaf extract does not impact on biofilm phase where the growth rate is very low. After that, at growth curves, but does induce biofilm dispersion in C. 16 h the number of viable cells was similar to the other albicans and interfere with AI2 treatments. A CDC reactor was used as the laboratory scale model The effects of 10 mg/l of E. acoroides leaf extract on system to grow a complex and mature C. albicans biofilm the cellular communication of V. harveyi were reported in the absence and presence of 0.01 mg/l E. acoroides leaf in Fig. 9. The results highlighted a significant increase in extract, the most effective concentration obtained from the relative luminescence emitted at time 8 h compared the adhesion assay. to the control (25.75 ± 7.49%). Results in Fig. 7a indicated a significant reduction in the number of viable cells adhered on coupon surfaces treated Discussion with the extract, compared to the untreated ones, after Biofilm resistance to antimicrobial agents is a major 24 h (reduction of fungal coverage up to 26.77 ± 9.01%). worldwide health care issue. Therefore, a successful re- Coupons collected after 48 and 72 h showed no significant duction of surface colonization can be a potential strat- differences between the treated biofilm and the control. egy for the management of unwanted biofilms, especially A significant increase in the number of dispersed on medical devices and work surfaces. cells in the treated biofilm (70 ± 6.83%) was observed In this context, the use of plant-derived extracts to (Fig. 7b). modulate biofilm genesis and dispersion may be a viable A colony biofilm assay was used to grow a complex alternative. The present study is the first report describ- and mature E. coli biofilm in the presence and absence ing the antibiofilm efficacy of non-lethal concentrations of 10 mg/l E. acoroides. Results in Fig. 8 showed no sig- of E. acoroides, H. pinifolia and H. ovalis methanol nificant reduction in the number of viable cells during extracts in counteracting microbial biofilms, highlighting biofilm formation on the membrane treated with the the possibility that the selected seagrass species act as an extract, compared to the untreated, after 18 h in all the extracellular signal mediating their biofilm activities. experimental conditions. Treatment 3 showed a growth E. coli and C. albicans were chosen as model systems rate slowdown in the interval 6–8, in which E. coli cells for bacterial and fungal infections, respectively. E. coli De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 8 of 17 Table 2 Individual compounds identified by comparison of MS/MS spectra with database entries in Enhalus acoroides leaf extract (A), E. acoroides root extract (B), Halophila ovalis leaf extract (C) and Halodule pinifolia leaf extract (D) No RT Mass MS/MS Name Accession Source A- E. acoroides leaf extract 1 2.5 343.03 201.02, 157.03, 59.01 n. i. –– 2 2.7 312.12 179.05, 132.06, 89.02 n. i. –– 3 3.3 367.1 277.07, 187.04, 157.03 n. i. –– 4 7.2 134.04 107.03, 92.02 Adenine PT200393 ReSpect 5 13.7 637.1 461.07, 285.04 Kaempferol-3-glucuronide, mod. PT209240 ReSpect 6 14.8 275.15 233.12, 119.05 n. i. –– 7 15.2 121.03 92.02, 77.03 Benzoic acid KO000321 MassBank 8 18.6 527.02 285.04, 241.00, 96.96 n. i. –– 9 20.1 511.05 269.04, 241.00, 96.96 n. i. –– 10 20.8 187.09 169.08, 125.09, 97.06 Azelaic acid KO000124 MassBank 11 21.3 447.09 285.04 Kaempferol-3-O-glucoside PS042209 ReSpect 12 22.5 461.07 285.04 Kaempferol-3-glucuronide PS092408 ReSpect 13 27.5 285.04 151.00, 133.03 Luteolin PS040410 ReSpect 14 29.5 269.04 225.05, 151.00, 117.03 Apigenin PT203930 ReSpect B- E. acoroides root extract 1 2.4 343.03 201.02, 157.03, 59.01 n. i. –– 2 2.7 312.12 179.05, 132.06, 89.02 n. i. –– 3 2.9 377.08 341.11, 179.05, 119.03, 89.02 Galactinol dihydrate, mod. PT211910 ReSpect 4 4.3 216.98 173.02, 156.98, 136.94, 59.01 n. i. –– 5 7.2 134.04 107.03, 92.02 Adenine PT200393 ReSpect 6 9.6 577.12 451.10, 425.08, 407.07, 289.07, 125.02 Procyanidin B2 PT204580 ReSpect 7 12.3 289.07 245.07, 203.07, 151.04, 109.03 +(−) Epicatechin PT204560 ReSpect 8 13.8 637.1 461.07, 285.04 Kaempferol-3-glucuronide, mod. PT209240 ReSpect 9 14.0 469.08 275.02, 193.05, 178.02, 149.06, 96.96 n. i. –– 10 14.8 275.15 233.12, 119.05 n. i. –– 11 15.3 121.03 92.02, 77.03 Benzoic acid KO000321 MassBank 12 20.8 187.09 169.08, 125.09, 97.06 Azelaic acid KO000124 MassBank 13 22.6 461.07 285.04 Kaempferol-3-glucuronide PS092408 ReSpect 14 24.1 299.05 284.03, 256.03, 133.03 Kaempferide PT204030 ReSpect 15 27.5 285.04 151.00, 133.03 Luteolin PS040410 ReSpect 16 29.5 269.04 225.05, 151.00, 117.03 Apigenin PT203930 ReSpect 17 31.2 329.23 229.14, 211.13, 171.10 n. i. –– C- H. ovalis leaf extract 1 2.4 343.03 201.02, 157.03, 59.01 n. i. –– 2 2.9 377.08 341.11, 179.05, 119.03, 89.02 Galactinol dihydrate, mod. PT211910 ReSpect 3 4.3 216.98 173.02, 156.98, 136.94, 59.01 n. i. –– 4 13.3 261.04 217.05, 189.05, 133.02 n. i. –– 5 15.5 121.03 92.02, 77.03 Benzoic acid KO000321 MassBank 6 16.3 306.17 288.16 n. i. –– 7 17.5 479.08 316,02 Myricetin-3-galactoside PS092809 ReSpect 8 19.5 463.09 301,03 Quercetin-3-O-beta-D-galactoside PS046509 ReSpect 9 20.8 187.09 169.08, 125.09, 97.06 Azelaic acid KO000124 MassBank De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 9 of 17 Table 2 Individual compounds identified by comparison of MS/MS spectra with database entries in Enhalus acoroides leaf extract (A), E. acoroides root extract (B), Halophila ovalis leaf extract (C) and Halodule pinifolia leaf extract (D) (Continued) No RT Mass MS/MS Name Accession Source 10 21.1 317.02 271.02, 149.02 n.i. –– 11 21.3 447.09 285.04 Kaempferol-3-O-glucoside PS042209 ReSpect 12 23.5 301.03 255.03, 165.02, 133.03 n.i. –– 13 24.1 299.05 284.03, 256.03, 133.03 Kaempferide PS040309 ReSpect 14 25.7 285.04 239.03, 185.06, 143.05, 117.03 Kaempferol PR040027 MassBank 15 27.5 285.04 285.04, 151.00,133.02 Luteolin PT204043 ReSpect 16 29.4 269.04 225.05, 151.00, 117.03 Apigenin PT203930 ReSpect D- H. pinifolia leaf extract 1 2.4 343.03 201.02, 157.03, 59.01 n. i. –– 2 2.9 377.08 341.11, 179.05, 119.03, 89.02 Galactinol dihydrate, mod. PT211910 ReSpect 3 4.3 216.98 173.02, 156.98, 136.94, 93.03, 59.01 n. i. –– 4 6.6 473.07 311.04, 293.03, 179.03, 149.01 n. i. –– 5 9.6 577.12 451.10, 425.08, 407.07, 289.07, 125.02 Procyanidin B2 PT204580 ReSpect 6 12.1 289.07 245.07, 203.07, 151.04, 109.03 +(−) Epicatechin PT204560 ReSpect 7 14.0 469.08 275.02, 193.05, 178.02, 149.06, 96.96 n. i. –– 8 19.1 641.17 473.13, 311.07, 167.03 n. i. –– 9 19.7 549.09 505.10, 463.09, 300.02, 271.02, 255.02 Quercetin-3-(6-malonyl)-glucoside PT209340 ReSpect 10 20.8 187.09 169.08, 125.09, 97.06 Azelaic acid KO000124 MassBank 11 21.1 505.09 463.08, 300.02, 271.02 Quercetin-3-O-beta-D-galactoside, mod. PT204650 ReSpect 12 21.8 463.08 300.03, 271.02 Quercetin-3-O-beta-D-galactoside PT204650 ReSpect 13 22.4 433.07 300.02, 271.02, 255.03, 179.00 Quercetin-3-arabinoside PT209320 ReSpect 14 23.4 447.09 284.03, 255.03, 227.03 Kaempferol-3-glucoside PT209270 ReSpect 15 24.6 417.08 284.03, 255.03, 227.03 Kaempferol-3-O-alpha-L-arabinoside PT209220 ReSpect 16 26.3 301.03 178.99, 151.00, 121.03, 107.01 Quercetin PT204090 ReSpect 17 27.4 285.04 199.03, 175.04, 151.00, 133.02 Luteolin PT204043 ReSpect 18 27.7 315.05 300.02, 271.02, 255.03 Isorhamnetin PM007432 ReSpect 19 29.5 269.04 225.05, 151.00, 117.03 Apigenin PT203930 ReSpect No = number of peak in Fig. 9, RT = retention time, Mass = mass of precursor ion, MS/MS = fragment spectra obtained at − 30 eV, Accession = accession number in database, Source = database used, n. i. = not identified, mod. = modified biofilms are found to be the major causative agent of antibacterial activity of H. ovalis and H. pinifolia extracts, many intestinal infections, for recurrent urinary tract in- obtained using different solvents, against different micro- fections, and it also responsible for indwelling medical bial strains, recording maximum antibacterial activity by device-related infectivity [43]. C. albicans is one of the the ethanol extract of H. pinifolia. Instead, Choi et al. [48] very few fungal species causing disease in humans. These reported the antimicrobial properties of Zostera marina infections range from superficial mucosal and dermal methanol extract and its organic solvent fractions on three infections, such as thrush, vaginal yeast infections, and human skin pathogens (Staphylococcus aureus, S. epider- diaper rash, to vascular catheters and dental implants midis and C. albicans), and Natrah et al. [47]reportedthe infections [44]. antibacterial properties of methanol extracts of E. acor- The bioactive properties of the seagrass species selected oides and other seagrass and seaweed species on different in this work are well known, and have been reported in aquaculture pathogens (Aeromonas hydrophila, Vibrio detail by several authors [45–47]. However, until now alginolyticus, V. parahaemolyticus, V. anguillarum and attention has mainly focused on the antimicrobial activity others). of seagrass extracts, which, through disk diffusion assays, In contrast, to the best of our knowledge, no papers were investigated not in their capacity as biofilm-forming have investigated the antibiofilm activity of Enhalus microorganisms but in their planktonic state. Using lethal acoroides, Halodule pinifolia and Halophila ovalis at concentrations, Umamaheshwari et al. [46]reported the non-lethal concentrations against bacterial (E. coli)and De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 10 of 17 Fig. 3 E. coli (a) and C. albicans (b) planktonic growth without (positive control) and with each seagrass extract at 100 ppm. The positive control was set up with mineral medium supplemented with glucose at 5 g/l. Stars indicate statistically significant differences (Tukey’s HSD, p ≤ 0.05) between the means of three independent replicates. (EAL = Enhalus acoroides leaf; EAR = Enhalus acoroides root; HPL = Halodule pinifolia leaf; HOL = Halophila ovalis leaf; C + = Positive control) Fig. 4 OD-based growth curves of C. albicans in absence (positive control) and in presence of each seagrass extract at 10 and 100 ppm. Maximum specific growth rate (μ )and the goodnessof fit (R ) obtained by the Gompertz model. Data represent the mean ± SDs of three independent measurements. Means reported showed no statistically significant differences between the positive control and treated samples (Tukey’sHSD, p ≥ 0.05). (EAL = Enhalus acoroides leaf; EAR = Enhalus acoroides root; HPL = Halodule pinifolia leaf; HOL = Halophila ovalis leaf; C + = Positive control) De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 11 of 17 Fig. 5 OD-based growth curves of E. coli in absence (positive control) and in presence of each seagrass extract at 10 and 100 ppm. Maximum specific growth rate (μ )and the goodnessof fit (R ) obtained by the Gompertz model. Data represent the mean ± SDs of three independent measurements. Means reported showed no statistically significant differences between the positive control and treated samples (Tukey’sHSD, p ≥ 0.05). (EAL = Enhalus acoroides leaf; EAR = Enhalus acoroides root; HPL = Halodule pinifolia leaf; HOL = Halophila ovalis leaf; C + = Positive control) fungal (C. albicans) biofilms. To this end, methanol ex- biofilm formation, microtiter based assays were per- tracts, obtained from different organs of three seagrass formed. The results revealed excellent anti-adhesion ac- species (namely, Enhalus acoroides leaves and roots, tivity for E. acoroides leaf extract, reducing fungal Halophila ovalis leaves and Halodule pinifolia leaves) coverage up to 74% and bacterial coverage up to 61% at were screened for their ability to modulate biofilm gen- 0.01 and 10 mg/l, respectively. Therefore, 0.01 mg/l and esis without killing cells. Methanol was used as the 10 mg/l E. acoroides leaf extract were chosen as the extraction solvent, having been previously reported as best non-biocidal concentrations for C. albicans and E. the most effective solvent to obtain high concentra- coli respectively, and were used in the subsequent stud- tions of bioactive compounds with antibacterial activity ies. These concentrations significantly decreased the from seagrasses, compared to other extraction solvents number of adhered cells on a hydrophobic surface, [45, 49, 50]. more so than on the hydrophilic one. Previous studies Before evaluating the antibiofilm activity, the extracts, had highlighted the preference for hydrophobic sur- at concentrations of 100 mg/l, were first proved to not faces, these reporting a decreased adhesion on the act as a carbon and energy source nor to affect the hydrophobic surface compared to the hydrophilic [51, cellular growth of C. albicans and E. coli.Therefore, 52]. This is probably due to the hydrophobic nature of concentrations ≤100 mg/l plant extract were used in the aerial surfaces of plants [53]. the subsequent studies. In the present study the anti-adhesion activity of the With the aim of investigating the effects of seagrass seagrass extracts was dose-dependent, but the highest extracts on cell adhesion to surfaces, the first step of concentrations did not correspond to those with the De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 12 of 17 Fig. 6 Microplate-based biofilm assay. Percentage reduction of the number of adhered cells of E. coli and C. albicans on hydrophilic and hydrophobic surface in presence of non-lethal concentrations of seagrass extracts. According to post hoc analysis (Tukey’s HSD, p ≤ 0.05), stars indicate statistically significant differences between the means of three independent replicates. In addition, the mean ± SDs of the percentage reduction of the number of adhered cells with seagrass extracts at non-lethal concentrations on hydrophilic and hydrophobic surface are reported in the table. The higher anti-adhesion effect for each microorganism was highlighted. (EAL = Enhalus acoroides leaf; EAR = Enhalus acoroides root; HPL = Halodule pinifolia leaf; HOL = Halophila ovalis leaf) best performance. Indeed, several studies have reported literature, is defined as hormesis, an adaptive behavior a weak activity of the compounds at low and high of microorganisms to provide resistance to environ- concentrations, and excellent activity at intermediate mental stress and improve the allocation of resources concentrations [54]. Such a response, widely known in to ensure cell stability [19, 55]. De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 13 of 17 Fig. 7 CDC biofilm growth on polycarbonate coupons (a) and biofilm dispersion rate (b)of C. albicans in absence (positive control) and in presence (treated) of 0.01 ppm of Enhalus acoroides leaf extract. Stars indicate statistically significant differences (Tukey’s HSD, p ≤ 0.05) between the means of three independent replicates. (C + = Positive control; EAL = Enhalus acoroides leaf) To further explore the effect of the most promising the number of dispersed cells in the treated biofilm, seagrass extract on biofilm development and detachment, compared with the untreated (70 ± 6.83%), suggesting CDC reactors were employed to reproduce biofilm at the a further mechanism of action for the seagrass extract solid/liquid interface, while for the assessment of the anti- as biofilm dispersing agent. In fact, the phase of bio- biofilm effect in the adhesion phase microplate-based film dispersion could be an interesting target for the biofilm assays are the most suitable [41, 56, 57]. In this development of new antibiofilm strategies, forcing the study, a significant reduction in fungal coverage (up to planktonic state and reestablishing the efficacy of 26.77 ± 9.01%) after 24 h (static adhesion phase) was traditional antimicrobial agents [4, 58]. Literature with observed in presence of 0.01 mg/l E. acoroides leaf information related to C. albicans biofilm dispersion is extract. This result confirms the anti-adhesion activity scarce. Farnesol and cis-2-decenoic acid showed observed in microtiter assays. Coupons collected after dispersion-promotion of microcolonies of C. albicans 48 and 72 h showed no significant differences between biofilm [58, 59]. In addition, Villa et al. [60]reported that treated and control samples. non-lethal concentrations of Muscari comosum ethanol In order to assess the possibility of 0.01 mg/l E. acoroides bulb extract can modulate yeast adhesion and subsequent leaf extract to promote C. albicans biofilm-detachment biofilm development on abiotic surfaces, and such con- from the surface of coupons, a biofilm dispersion assay centrations could provide an extracellular signal respon- was performed. Results showed a significant increase in sible for biofilm dispersion. Fig. 8 Biofilm growth at the solid/air interface. E. coli biofilm grown on polycarbonate membrane under three experimental conditions: i) treatment 1: growth in contact with 1 ml of LB with 10 ppm of E. acoroides leaf extract; ii) treatment 2: overnight culture grown with 10 ppm of E. acoroides leaf extract and then growth in contact with 1 ml of LB; iii) treatment 3: overnight culture grown with 10 ppm of E. acoroides leaf extract and growth in contact with 1 ml of LB with 10 ppm of E. acoroides leaf extract. In the positive control, microorganisms grew in 1 ml LB inside a basolateral well without the extract. Data obtained were divided by the area of the membrane, and means were reported. The experiment was repeated three times. (T1 = treatment 1; T2 = treatment 2; T3 = treatment 3; C + = Positive control) De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 14 of 17 Fig. 9 Relative luminescence emitted by Vibrio harveyi in absence (positive control) and in presence of 10 ppm of E. acoroides leaf extract for 24 h. The relative luminescence has been calculated by normalizing luminescence by the number of adhered cells. Stars indicate statistically significant differences (Tukey’sHSD,p ≤ 0.05) between the means of three independent replicates. (C + = Positive control; EAL = Enhalus acoroides leaf) For E. coli, the CDC reactor was not suitable to evalu- phenolic compound occurring in the seagrass Zostera ate the possible effects of the extracts on the biofilm marina. In fact, it has been hypothesized that the stages. Also other authors have reported the poor biofilm accumulation of AI-2 above a threshold level leads to formation exhibited by E. coli K-12 strain under hydro- reduced biofilm formation due to the induction of a dynamic conditions [61–63]. The effect of 10 mg/l of E. hypermotile phenotype that is unable to adhere to the acoroides leaf extract on E. coli biofilm formation was then surface [64]. Huber et al. [65] demonstrated that some evaluated using a membrane-supporting biofilm reactor, polyphenolic compounds containing a gallic acid residue which allowed the formation of a biofilm at the solid/air commonly produced by some plant species inhibited interface. This technique forced the cells to attach to a sur- intercellular communication in bacteria. Truchado et al. face, a feature that allowed direct investigation of the effect [66] reported the ability of some phytochemical com- of the selected extract on the development of the biofilm, pounds (cinnamaldehyde, ellagic acid, resveratrol, rutin whilst bypassing the effect on the adhesion phase. and pomegranate extract) to interfere with the quorum No significant reduction in the number of viable cells sensing system of Yersinia enterocolitica and Erwinia during biofilm formation on the membrane treated with cartovora. the extract, compared to the untreated, after 18 h in all It has been well known that the antibiofilm activity of the experimental conditions was observed. Treatment 3 plant extracts is closely linked with the content of sec- showed a growth rate slowdown in the interval 6–8h,in ondary metabolites, such as phenols and/or flavonoids, which E. coli cells were in contact with the extract dur- which represent the total amount of phenolic com- ing both overnight growth and biofilm formation (reduc- pounds in a plant extract [13]. The phenolic compound tion of cellular growth, compared to the control, up to content is also deeply associated with the antioxidant 48.64 ± 4.02%). Interestingly, treatment 3 showed a bi- activity of plant extracts [67]. Therefore, we determined phasic growth curve compared with the growth curves the total phenolic acid (TPC) and flavonoid (TFC) content of the other treatments, a trend that could be explained and the antioxidant activity (ORAC) of methanolic ex- by the bioluminescence produced by V. harvey. As sig- tracts in order to highlight features of the most promising naling molecules play an important role in biofilm devel- antibiofilm extract, the E. acaroides leaf extract. Results opment and detachment, the effects of 10 mg/l of E. show that E. acaroides leaf extract presents the lower TPC acoroides leaf extract were investigated using V. harveyi, and TFC values compared to other seagrasse extracts. suggesting other possible antibiofilm mechanisms of ac- Although the low content of phenolic compounds, the E. tion of compounds in the chosen seagrass extract. The acaroides leaf extract displays a higher ORAC value com- results revealed that at time 8 h, the samples treated pared to the root extract. This indicates the abundance of with the leaf extract showed a significant increase in the other, non-phenolic compounds with antioxidant capacity relative luminescence emitted, compared to the control in the leaves of E. acoroides. Cattò et al. [39] suggested the (25.75 ± 7.49). Villa et al. [64] reported an increase of importance of antioxidant compounds in hindering autoinducer-2 (AI-2) activity and a reduction in biofilm biofilm formation. The researcher discovered that the formation in E. coli cells treated with zosteric acid, a mechanism of action behind the antibiofilm performance De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 15 of 17 of zosteric acid, a secondary metabolite of the seagrass Acknowledgments We would like to thank Dr. Vy Xuan Nguyen for the identification and Zostera marina, is related to the antioxidant activity of the collection of the seagrass samples in Vietnam used in this work. molecule, and its interaction with the WrbA protein re- sponsible maintaining cellular homeostasis and defense Funding This work was supported by the German-Italian bilateral project “Bioactive against oxidative stress. secondary compounds from halophyte species inhibit biofilm formation of To gain more insight into possible antibiofilm com- plant-pathogenic microorganisms on plant surfaces” (SAB-HAL), MIUR-DAAD pounds in the seagrass extracts, individual substances in Joint Mobility Program, ID-57265315. the methanolic extract were analyzed by LC-MS. Prelim- Availability of data and materials inary analysis shows that the phytochemical profile of The datasets used and/or analyzed during the current study available from the E. acaroides leaf extract is mainly characterized by the corresponding author on reasonable request. the presence of the flavones apigenin and luteolin, three Authors’ contributions kaempferol derivates and the carboxylic acids benzoic DVL performed the biological experiments and wrote the manuscript. and azelaic acid. This unique quantitative and qualitative GY extracted and analyzed the crude extracts from the plants by MS. CC participated in the design of the study and provided technical advices and lab chemical composition confers antibiofilm properties to supports. VF conceived, designed and coordinated the study. VF contributed the E. acaroides leaf extract. substantially to the writing and revising of the manuscript. CF and PJ Some of these compounds have shown to exhibit anti- participated in the design of the study, in discussions and reviewed the manuscript. All authors read and approved the final manuscript. biofilm properties at non-lethal concentrations. Kaemp- ferol, apigenin and luteolin from red wine reduced Competing interest biofilm formation of methicillin-sensitive S. aureus sig- The authors declare that they have no competing interests. nificantly [68]. Sánchez et colleagues [69] reported that Ethics approval and consent to participate sub-lethal concentrations of plant extracts inhibit E. coli Not applicable. and S. aureus biofilms. The antibiofilm properties of the extracts were associated to the presence of flavonoids, Publisher’sNote such as kaempferol and apigenin, which modulate bac- Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. terial cell-cell communication by suppressing the activity of the autoinducer-2 [70]. However, we should keep in Author details mind that the antibiofilm effects of plant extracts could Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, Università degli Studi di Milano, via Celoria 2, 20133 Milan, Italy. Institute of be the result of interactions among different compo- Botany, Leibniz University Hannover, Herrenhäuserstr. 2, D-30419 Hannover, nents of the extract at specific concentrations, and not Germany. only due to the effects of a single, predominant com- Received: 16 December 2017 Accepted: 15 May 2018 pound [4, 71]. Conclusions References 1. Costerton JW. Introduction to biofilm. Int J Antimicrob Agents. 1999;11:217–21. In conclusion, the E. acoroides leaf extract proved to be 2. Hall-Stoodley L, Costerton WJ, Stoodley P. Bacterial biofilms: from the the most promising extract among those tested. Indeed, natural environment to infectious diseases. Nat Rev Microbiol. 2004;2:95– the selected non-lethal concentrations of E. acoroides 108. https://doi.org/10.1038/nrmicro821. 3. Rayner J, Veeh R, Flood J. Prevalence of microbial biofilms on selected fresh leaf extract were found to exert an antibiofilm effect on produce and household surfaces. Int J Food Microbiol. 2004;95:29–39. C. albicans and E. coli biofilm in the first phase of bio- 4. Villa F, Villa S, Gelain A, Cappitelli F. Sub-lethal activity of small molecules film genesis, opening up the possibility of developing from natural sources and their synthetic derivatives against biofilm forming nosocomial pathogens. Curr Top Med Chem. 2013;13(24):3184–204. preventive strategies to hinder the adhesion of micro- 5. Macià MD, Rojo-Molinero E, Oliver A. Antimicrobial susceptibility testing in bial cells to surfaces. The leaf extract also affected the biofilm-growing bacteria. Clin Microbiol Infect. 2014;20(10):981–90. https:// dispersion and maturation steps in C. albicans and E. doi.org/10.1111/1469-0691.12651. 6. Percival SL, Suleman L, Vuotto C, Donelli G. Healthcare-associated infections, coli respectively, suggesting an important role in cell medical devices and biofilms: risk. tolerance and control J Med Microbiol. signaling processes. These effects could be explained by 2015;64(Pt 4):323–34. https://doi.org/10.1099/jmm.0.000032. the presence of active compounds like kaempferol and 7. Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC. Hidden killers: human fungal infections. Sci Transl Med. 2012;4:165rv113. https://doi. apigenin at specific concentrations in the extracts of E. org/10.1126/scitranslmed.3004404. acoroides, which are known to possess biofilm inhibit- 8. Akbari F, Kjellerup BV. Elimination of bloodstream infections associated with ing properties. Furthermore, there could be a synergis- Candida albicans biofilm in intravascular catheters. Pathogens 2015;4(3):457– 69. https://doi.org/10.3390/pathogens4030457. tic action of these flavonoids with other compounds 9. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. occurring in the plant, enhancing the global antibiofilm Lancet. 2001;358:135–8. https://doi.org/10.1016/S0140-6736(01)05321-1. effect. Currently, the leaf extract is being investigated 10. Smith K, Hunter IS. Efficacy of common hospital biocides with biofilms of multi-drug resistant clinical isolates. J Med Microbiol. 2008;57:966–73. with the objective of testing fractions for identifying the https://doi.org/10.1099/jmm.0.47668-0. active compounds and to better understand the mecha- 11. Lebeaux D, Ghigo JM, Beloin C. Biofilm-related infections: bridging the gap nisms of action of this seagrass species. between clinical management and fundamental aspects of recalcitrance De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 16 of 17 toward antibiotics. Microbiol Mol Biol Rev. 2014;78(3):510–43. https://doi. 32. Dewanto V, Wu X, Adom KK, Liu RH. Thermal processing enhances the org/10.1128/MMBR.00013-14. nutritional value of tomatoes by increasing total antioxidant activity. J Agric 12. Chung PY. Plant-derived compounds as potential source of novel anti- Food Chem. 2002;50:3010–4. https://doi.org/10.1021/jf0115589. biofilm agents against Pseudomonas aeruginosa. Curr Drug Targets. 2017; 33. Huang D, Ou B, Hampsch-Woodill M, Flanagan JA, Prior RL. High- 18(4):414–20. https://doi.org/10.2174/1389450117666161019102025. throughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate 13. Choi HA, Cheong DE, Lim HD, Kim WH, Ham MH, Oh MH, Wu Y, Shin HJ, fluorescence reader in 96-well format. J Agric Food Chem. 2002;50(16): Kim GJ. Antimicrobial and anti-biofilm activities of the methanol extracts of 4437–44. https://doi.org/10.1021/jf0201529. medicinal plants against dental pathogens Streptococcus mutans and Candida albicans. J Microbiol Biotechnol. 2017;27(7):1242–8. https://doi.org/ 34. Gillespie KM, Chae JM, Ainsworth EA. Rapid measurement of total 10.4014/jmb.1701.01026. antioxidant capacity in plants. Nat Protoc. 2007;2(4):867–70. https://doi.org/ 14. da Silva DT, Herrera R, Batista BF, Heinzmann BM, Labidi J. Physicochemical 10.1038/nprot.2007.100. characterization of leaf extracts from Ocotea lancifolia and its effect against 35. Horai H, Arita M, Kanaya S, Nihei Y, Ikeda T, Suwa K, Ojima Y, wood-rot fungi. Int Biodeterior Biodegradation. 2017;117:158–70. doi.org/10. Tanaka K, Tanaka S, Aoshima K, Oda Y, Kakazu Y, Kusano M, Tohge T, 1016/j.ibiod.2016.12.007 Matsuda F, Sawada Y, Yokota Hirai M, Nakanishi H, Ikeda K, Akimoto N, 15. Teanpaisan R, Kawsud P, Pahumunto N, Puripattanavong J. Screening for Maoka T, Takahashi H, Ara T, Sakurai N, Suzuki H, Shibata D, Neumann S, antibacterial and antibiofilm activity in Thai medicinal plant extracts against Iida T, Tanaka K, Funatsu K, Matsuura F, Soga T, Taguchi R, Saito K, oral microorganisms. J Tradit Complement Med. 2017;7(2):172–7. doi.org/10. Nishioka T. MassBank: a public repository for sharing mass spectral data 1016/j.jtcme.2016.06.007 for life sciences. J Mass Spectrom. 2010;45:703–14. https://doi.org/10. 16. Abiala M, Olayiwola J, Babatunde O, Aiyelaagbe O, Akinyemi S. Evaluation of 1002/jms.1777. therapeutic potentials of plant extracts against poultry bacteria threatening 36. Sawada Y, Nakabayashi R, Yamada Y, Suzuki M, Sato M, Sakata A, Akiyama K, public health. BMC Complement Altern Med. 2016;16(1):417. Sakurai T, Matsuda F, Aoki T, Hirai MY, Saito K. RIKEN tandem mass spectral database (ReSpect) for phytochemicals: a plant-specific MS/MS-based data 17. Bisi-Johnson MA, Obi CL, Samuel BB, Eloff JN, Okoh AI. Antibacterial activity resource and database. Phytochemistry. 2012;82:38–45. https://doi.org/10. of crude extracts of some south African medicinal plants against multidrug 1016/j.phytochem.2012.07.007. resistant etiological agents of diarrhoea. BMC Complement Altern Med. 37. Zwietering MH, Jongenburger I, Rombouts FM, van’t Riet K. Modeling of the 2017;17(1):321. https://doi.org/10.1186/s12906-017-1802-4. bacterial growth curve. Appl Environ Microbiol 1990;56:1875–1881. 18. Elisha IL, Botha FS, McGaw LJ, Eloff JN. The antibacterial activity of extracts of nine plant species with good activity against Escherichia coli against five 38. Villa F, Albanese D, Giussani B, Stewart P, Daffonchio D, Cappitelli F. other bacteria and cytotoxicity of extracts. BMC Complement Altern Med. Hindering biofilm formation with zosteric acid. Biofouling. 2010;26:739–52. 2017;17(1):133. https://doi.org/10.1186/s12906-017-1645-z. https://doi.org/10.1080/08927014.2010.511197. 19. Villa F, Cappitelli F. Plant-derived bioactive compounds at sub-lethal 39. Cattò C, Dell’Orto S, Villa F, Villa S, Gelain A, Vitali A, Marzano V, Baroni S, concentrations: towards smart biocide-free antibiofilm strategies. Forlani F, Cappitelli F. Unraveling the structural and molecular basis Phytochem Rev. 2013;12:245–54. https://doi.org/10.1007/s11101-013-9286-4. responsible for the anti-biofilm activity of zosteric acid. PLoS One. 2015; 10(7):e0131519. https://doi.org/10.1371/journal.pone.0131519. 20. Flowers TJ, Colmer TD. Salinity tolerance in halophytes. New Phytol. 2008; 40. Villa F, Pitts B, Stewart PS, Giussani B, Roncoroni S, Albanese D. 179:945–63. https://doi.org/10.1111/j.1469-8137.2008.02531.x. Efficacy of zosteric acid sodium salt on the yeast biofilm model 21. Flowers TJ, Colmer TD. Plant salt tolerance: adaptations in halophytes. Ann Candida albicans. Microb Ecol. 2011;62:584–98. https://doi.org/10.1007/ Bot. 2015:115327–31. https://doi.org/10.1093/aob/mcu267. s00248-011-9876-x. 22. Joshi R, Ramanarao MV, Bedre R, Sanchez L, Pilcher W, Zandkarimi H, Baisakh N. Salt adaptation mechanisms of halophytes: improvement of salt 41. Cattò C, Grazioso G, Dell’Orto S, Gelain A, Villa S, Marzano V, Vitali A, Villa F, tolerance in crop plants. In: Pandey GK, editor. Elucidation of abiotic stress Cappitelli F, Forlani F. The response of Escherichia coli biofilm to salicylic acid. signaling in plants. New York, NY: Springer; 2015. p. 243–79. https://doi.org/ Biofouling. 2017;33:235–51. https://doi.org/10.1080/08927014.2017.1286649. 10.1007/978-1-4939-2540-7_9. 42. Garuglieri E, Meroni E, Cattò C, Villa F, Cappitelli F, Erba D. Effects of sub- lethal concentrations of silver nanoparticles on a simulated intestinal 23. Selmar D. Potential of salt and drought stress to increase pharmaceutical prokaryotic-eukaryotic interface. Front Microbiol. 2018;8:2698. https://doi. significant secondary compounds in plants. Landbauforschung. 2008;58: org/10.3389/fmicb.2017.02698. 139–44. 43. Sharma G, Sharma S, Sharma P, Chandola D, Dang S, Gupta S, Gabrani R. 24. Ksouri R, Ksouri WM, Jallali I, Debez A, Magné C, Hiroko I, Abdelly C. Escherichia coli biofilm: development and therapeutic strategies. J Appl Medicinal halophytes: potent source of health promoting biomolecules Microbiol. 2016;121(2):309–19. https://doi.org/10.1111/jam.13078. Epub with medical, nutraceutical and food applications. Crit Rev Biotechnol. 2012; 2016 Mar 11 32:289–326. https://doi.org/10.3109/07388551.2011.630647. 25. Boestfleisch C, Wagenseil NB, Buhmann AK, Seal CE, Wade EM, Muscolo A, 44. Nobile CJ, Johnson AD. Candida albicans biofilms and human disease. Annu Papenbrock J. Manipulating the antioxidant capacity of halophytes to Rev Microbiol. 2015;69:71–92. https://doi.org/10.1146/annurev-micro- increase their cultural and economic value through saline cultivation. AoB 091014-104330. Plants. 2014;6:plu046. https://doi.org/10.1093/aobpla/plu046. 45. Kumar SC, Sarada DVL, Gideon TP, Rengasamy R. Antibacterial activity of 26. Hua KF, Hsu HY, Su YC, Lin IF, Yang SS, Chen YM. Study on the three south Indian seagrasses, Cymodocea serrulata, Halophila ovalis and antinflammatory activity of methanol extract from seagrass Zostera japonica. Zostera capensis. World J Microbiol Biotechnol. 2008;24(9):1989–92. https:// J Agric Food Chem. 2006;54:306–11. https://doi.org/10.1021/jf0509658. doi.org/10.1007/s11274-008-9695-5. 46. Umamaheshwari R, Thirumaran G, Anantharaman P. Potential antibacterial 27. Gokce G, Haznedaroglu MZ. Evaluation of antidiabetic, antioxidant and activities from vellar estuary; south east coast of India. Adv Biomed Res. vasoprotective effects of Posidonia oceanica extract. J Ethnopharmacol. 2009;3:140–3. 2008;115:122–30. 47. Natrah FMI, Harah ZM, Japar Sidik NI, Syahidah A. Antibacterial 28. Kannan RRR, Arumugam R, Anantharaman P. Chemical composition and activities of selected seaweed and seagrass from port Dickson coastal antibacterial activity of Indian seagrasses against urinary tract pathogens. Food water against different aquaculture pathogens. Sains Malays. 2015; Chem. 2012;135:2470–3. https://doi.org/10.1016/j.foodchem.2012.07.070. 29. Nguyen XV, Höfler S, Glasenapp Y, Thangaradjou T, Lucas C, Papenbrock J. 44(9):1269–73. New insights into the DNA barcoding of seagrasses. Syst Biodivers. 2015;13: 48. Choi HG, Lee JH, Park HH, Sayegh FAQ. Antioxidant and antimicrobial 496–508. https://doi.org/10.1080/14772000.2015.1046408. activity of Zostera marina L. extract. Algae. 2009;24(3):179–84. https://doi. org/10.4490/algae.2009.24.3.179. 30. Lucas C, Thangaradjou T, Papenbrock J. Development of a DNA barcoding 49. Lustigman B, Brown C. Antibiotic production by marine algae isolated from system for seagrasses: successful but not simple. PLoS One. 2012;7:e29987. the New York/New Jersey coast. Bull Environ Contam Toxicol. 1991;46:329– https://doi.org/10.1371/journal.pone.0029987. 35. https://doi.org/10.1007/BF01688928. 31. Dudonné S, Vitrac X, Coutière P, Woillez M, Mérillon JM. Comparative study 50. Sastry VMVS, Rao GRK. Antibacterial substances from marine algae: of antioxidant properties and total phenolic content of 30 plant extracts of successive extraction using benzene, chloroform and methanol. Bot Mar. industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J Agric 1994;37:357–60. https://doi.org/10.1515/botm.1994.37.4.357. Food Chem. 2009;57:1768–74. https://doi.org/10.1021/jf803011r. De Vincenti et al. BMC Complementary and Alternative Medicine (2018) 18:168 Page 17 of 17 51. Doss RP, Potter SW, Chastagner GA, Christian JK. Adhesion of nongerminated 70. Vikram A, Jayaprakasha GK, Jesudhasan PR, Pillai SD, Patil BS. Suppression of Botrytis cinerea conidia to several substrata. Appl Environ Microbiol. 1993;59: bacterial cell-cell signalling, biofilm formation and type III secretion system 1786–91. by citrus flavonoids. J Appl Microbiol. 2010;109(2):515–27. https://doi.org/10. 52. Amiri A, Cholodowski D, Bompeix G. Adhesion and germination of 1111/j.1365-2672.2010.04677.x. waterborne and airborne conidia of Penicillium expansum to apple and inert 71. Bazargani MM, Rohloff J. Antibiofilm activity of essential oils and plant surfaces. Physiol Mol Plant Pathol. 2005;67:40–8. https://doi.org/10.1016/j. extracts against Staphylococcus aureus and Escherichia coli biofilms. Food pmpp.2005.07.003. Control. 2016;61:156–64. https://doi.org/10.1016/j.foodcont.2015.09.036. 53. Koch K, Bhushan B, Barthlott W. Multifunctional surface structures of plants: an inspiration of biomimetics. Prog Mater Sci. 2009;54:137–78. https://doi. org/10.1016/j.pmatsci.2008.07.003. 54. Rickard AH, Palmer RJ, Blehert DS, Campagna SR, Semmelhack MF, Egland PG, Bassler BL, Kolenbrander PE. Autoinducer 2: a concentration-dependent signal for mutualistic bacterial biofilm growth. Mol Microbiol. 2006;60:1446– 56. https://doi.org/10.1111/j.1365-2958.2006.05202.x. 55. Calabrese EJ, Baldwin LA. Hormesis: the dose-response revolution. Annu Rev Pharmacol Toxicol. 2003;43:175–97. https://doi.org/10.1146/annurev. pharmtox.43.100901.140223. 56. Williams DL, Woodbury KL, Haymond BS, Parker AE, Bloebaum RD. A modified CDC biofilm reactor to produce mature biofilms on the surface of PEEK membranes for an in vivo animal model application. Curr Microbiol. 2011;62:1657–63. https://doi.org/10.1007/s00284-011-9908-2. 57. Coffey BM, Anderson GG. Biofilm formation in the 96-well microtiter plate. Methods Mol Biol. 2014;1149:631–41. https://doi.org/10.1007/978-1-4939- 0473-0_48. 58. Davies DG, Marques CN. A fatty acid messenger is responsible for inducing dispersion in microbial biofilms. J Bacteriol. 2009;191:1393–403. https://doi. org/10.1128/JB.01214-08. 59. Uppuluri P, Chaturvedi AK, Srinivasan A, Banerjee M, Ramasubramaniam AK, Köhler JR, Kadosh D, Lopez-Ribot JL. Dispersion as an important step in the Candida albicans biofilm developmental cycle. PLoS Pathog. 2010;6: e1000828. https://doi.org/10.1371/journal.ppat.1000828. 60. Villa F, Borgonovo G, Cappitelli F, Giussani B, Bassoli A. Sublethal concentrations of Muscari comosum bulb extract suppress adhesion and induce detachment of sessile yeast cells. Biofouling. 2012;28:1107–17. https://doi.org/10.1080/08927014.2012.734811. 61. Ghigo JM. Natural conjugative plasmids induce bacterial biofilm development. Nature. 2001;412:442–5. https://doi.org/10.1038/35086581. 62. Reisner A, Haagensen JA, Schembri MA, Zechner EL, Mølin S. Development and maturation of Escherichia coli K-12 biofilms. Mol Microbiol. 2003;48:933– 46. https://doi.org/10.1046/j.1365-2958.2003.03490.x. 63. Reisner A, Krogfelt KA, Klein BM, Zechner EL, Mølin S. 2006. In vitro biofilm formation of commensal and pathogenic Escherichia coli strains: impact of environmental and genetic factors. J Bacteriol. 2006;188:3572–81. https://doi. org/10.1128/JB.188.10.3572-3581.2006. 64. Villa F, Remelli W, Forlani F, Vitali A, Cappitelli F. Altered expression level of Escherichia coli proteins in response to treatment with the antifouling agent zosteric acid sodium salt. Environ Microbiol. 2012;14:1753–61. 65. Huber AB, Kolodkin AL, Ginty DD, Cloutier JF. Signaling at the growth cone: ligand-receptor complexes and the control of axon growth and guidance. Annu Rev Neurosci. 2003;26:509–63. https://doi.org/10.1146/annurev.neuro. 26.010302.081139. 66. Truchado P, Lopez-Galvez F, Gil MI, Tomas-Barberan FA, Allende A. Quorum sensing inhibitory and antimicrobial activities of honeys and the relationship with individual phenolics. Food Chem. 2009;115:1337–44. https://doi.org/10.1016/j.foodchem.2009.01.065. 67. Reis Giada ML. Food phenolic compounds: main classes, sources and their antioxidant power, oxidative stress and chronic degenerative diseases Jose Antonio Morales-Gonzalez, IntechOpen. 2013. https://doi.org/10.5772/51687. Available from: https://www.intechopen.com/books/oxidative-stress-and- chronic-degenerative-diseases-a-role-for-antioxidants/food-phenolic- compounds-main-classes-sources-and-their-antioxidant-power. 68. Cho HS, Lee JH, Cho MH, Lee J. Red wines and flavonoids diminish Staphylococcus aureus virulence with anti-biofilm and anti-hemolytic activities. Biofouling. 2015;31(1):1–11. https://doi.org/10.1080/08927014. 2014.991319. 69. Sánchez E, Rivas Morales C, Castillo S, Leos-Rivas C, García-Becerra L, Mizael Ortiz Martínez D. Antibacterial and antibiofilm activity of methanolic plant extracts against nosocomial microorganisms. J Evid Based Complementary Altern Med. 2016, Article ID 1572697;2016:8. https://doi.org/10.1155/2016/

Journal

BMC Complementary and Alternative MedicineSpringer Journals

Published: May 30, 2018

References

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off