Taxonomists find some plant genera challenging because of the few morphological differences or unclear characters among closely related species, which leads to the misidentification of taxa. DNA barcoding is an approach to identify species by using short orthologous DNA sequences, known as 'DNA barcodes'. Concatenated rbcL and matK sequences are considered DNA barcodes for seagrasses. However, these markers are not applicable to all members of seagrasses at the species level, especially within the genus Halophila. Our previous studies indicated that the internal transcribed spacer (ITS) showed higher species resolution than the concatenated rbcL and matK sequences in the case of Halophila ovalis and closely related species. In this study, 26 ITS, two rbcL and two matK consensus sequences from 18 seagrass taxa belonging to four families collected in India, Vietnam, Germany, Croatia and Egypt were processed. Molecular ITS analysis resolved five clades. The results also indicate that the Cymodoceaceae family might be a non-monophyletic group. In conclusion, ITS could be applied as a DNA barcode for seagrasses instead of the rbcL/matK system previously proposed.

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BackgroundBiofilms 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.MethodsMethanolic 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.ResultsIt 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.ConclusionsE. 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.

The extracts of two mangrove species, Bruguiera cylindrica and Laguncularia racemosa, have been analyzed at sub-lethal concentrations for their potential to modulate biofilm cycles (i.e., adhesion, maturation, and detachment) on a bacterium, yeast, and filamentous fungus. Methanolic leaf extracts were also characterized, and MS/MS analysis has been used to identify the major compounds. In this study, we showed the following. (i) Adhesion was reduced up to 85.4% in all the models except for E. coli, where adhesion was promoted up to 5.10-fold. (ii) Both the sum and ratio of extracellular polysaccharides and proteins in mature biofilm were increased up to 2.5-fold and 2.6-fold in comparison to the negative control, respectively. Additionally, a shift toward a major production of exopolysaccharides was found coupled with a major production of both intracellular and extracellular reactive oxygen species. (iii) Lastly, detachment was generally promoted. In general, the L. racemosa extract had a higher bioactivity at lower concentrations than the B. cylindrica extract. Overall, our data showed a reduction in cells/conidia adhesion under B. cylindrica and L. racemosa exposure, followed by an increase of exopolysaccharides during biofilm maturation and a variable effect on biofilm dispersal. In conclusion, extracts either inhibited or enhanced biofilm development, and this effect depended on both the microbial taxon and biofilm formation step.

In the search of new compounds with biofilm‐inhibiting properties, mangroves with their richness of secondary metabolites can be a valuable resource. Crude methanolic leaf extracts from the mangrove Laguncularia racemosa enriched in phenolic substances cause a reduction in initial cell adhesion of Candida glabrata and Candida albicans, but not on Escherichia coli. LC/MS‐guided fractionation of the phenolic compounds resulted in 19 fractions, of which ten were analyzed for their bioactivity against cell adhesion. Effects on cell adhesion and planktonic growth of Escherichia coli, Candida glabrata and Candida albicans were measured in 96‐well microtiter plates in the presence of 0.2 mg ml−1 of the isolated fractions. Two fractions caused a reduction of cell adhesion of Candida albicans. These fractions containing bioactive compounds were analyzed by LC/MS and NMR spectroscopy. Casuarinin and digalloyl‐hexahydroxydiphenoyl‐glucose were identified in the active fractions, in addition to three signals of ellagitannins. These results indicate a specific mode of action of hydrolysable tannins against cell adhesion of Candida albicans, which needs to be further analyzed.