The research of new strategies to inhibit adhesion and biofilm formation on surfaces immersed in seawater is based on the use of antiadhesive compounds without impact on the environment, so without bacteriostatic or bactericide effects. Hence, the efficient concentration inhibiting 50% of bacterial adhesion (EC50), the inhibitory concentration for 50% of the bacterial growth (IC50) and the lethal concentration for 50% of the bacteria (LC50) were determined.
2.4.2. Impact on Bacterial Adhesion
The anti-bioadhesion of the microalgal extracts were evaluated on the eight marine bacterial strains at a same concentration (50 μg mL−1
) in a dynamic mode (flow cell). For each microalgal extract, the inhibition percentages of bacterial adhesion were determined by comparison between the standard conditions (without extract) and in the presence of the extract. All results were summarized in Table 2
In this table, microalgae were ranked versus
their significant activity (p
< 0.05, post-hoc HSD Tukey) from the best to the poorest activity. Four groups were identified. Microalgae belonging to group a
have shown the highest bacterial inhibition. Three strains were in this group: P-78, P-43 and P-63 followed by P-60 (group b). The majority of extracts inhibited more than 50% bacterial adhesion of these strains. Microalgae classed in the group g showed the lowest activity: five strains were included in this group. The inhibition of bacterial adhesion was extremely low for most extracts. Other extracts had an intermediate activity (groups b–f).
For illustration, the Figure 4
showed the results obtained for the more active extract (P-78) against one bacterial strain of each origin (Atlantic Ocean, Indian Ocean and Mediterranean See).
To observe possible clusterization of the different extracts depending on their activities on all the bacterial strains, a principal component analysis (PCA) was realized. It was based on the capacity of microalgal extracts to inhibit bacterial adhesion. The resulting plots showed that the two first components of this statistical model accounted for 57.52% (38.02% for the first component axis and 19.50% for the second one) of the total variance of the dataset (Figure 5
). Thus, the first axis accounted for 38.02% and the second axis 19.50%. On the resulting score plot (Figure 5
A), the first dimension allowed a gross discrimination between dinoflagellates extracts (excepted for P-59), positively correlated with the first axis, and the major part of the other extracts (excepted C-59, P-59, and P-89), negatively correlated with the first axis. Moreover, by comparison with Table 2
, the most and the least active strains were situated positively and negatively on this first axis, respectively.
As noticed on the variable factor map, the first axis appeared to differentiate the anti-adhesion activity on most of the bacterial strains (Shewanella sp. PVV6, Shewanella sp. TC11, Pseudoalteromonas sp. TC8, Paracoccus sp. 4M6 and Pseudoalteromonas sp. 5M6), except Shewanella sp. MVV1 and Bacillus sp. 4J6 whose activities mostly explained the second axis. This indicates that except for the latter, most of the bacterial strains exhibited similar relative activity for the different microalgal extracts. Consequently, the first axis turned up to be a good proxy of the overall activity. Therefore, dinoflagellates were well clustered and separated from other less active strains.
Cyanobacteria and to a lesser extent diatoms are well known to be excellent sources of natural metabolites with antibacterial activity [36
]. However, in this work, these taxonomic families were less efficient than Dinoflagellates. For example, the Symbiodinium
sp. (P-78) extract inhibited the adhesion of Bacillus
sp. 4J6 (Atlantic Ocean), Shewanella
sp. MVV1 (Indian Ocean) and Pseudoalteromonas lipolytica
TC8 (Mediterranean Ocean) at 61, 76 and 52% respectively as illustrated in Figure 4
. This microalgal strain was the most active: the adhesion of seven bacterial strains out of the eight studied marine bacteria was inhibited (inhibition >50%) by its extract. Alone, the adhesion of TC11 was only inhibited at 40%. The three other strains (P-43, P-63 and P-60) corresponded to the genus Amphidinium
. P-43 extract inhibited by more than 50% the adhesion of six of the eight bacterial strains assayed, whereas P-63 and P-60 extracts were highly active (inhibition >50%) against five bacterial strains. The genus Amphidinium
belong to the taxonomic group of dinoflagellates. Dinoflagellates are known to be important sources of toxins such as macrolides, polyketides, polyols and polyether [39
]. For example, zooxanthellamide Cs and symbioimine were identified from Symbiodinium
]. Amphidinin G, Karatungiols A and B, Carteraol E were isolated from Amphidinium
]. These compounds have been proven to show a wide range of biological activities (e.g., anti-resorptive, anti-inflammatory, antifungal, antiprotozoal and antibacterial).
Bacteria showed various sensitivity in the presence of microalgal extracts (Table 3
sp. 4J6, Pseudoalteromonas
sp. 5M6, Shewanella
sp. PVV6 and Pseudoalteromonas lipolytica
TC8 were the more sensitive bacterial strains: their adhesion was inhibited by at least eleven microalgal extracts. Extracts acted indifferently against Gram-positive and Gram-negative bacteria, although Gram positive bacteria are known to show higher sensitivity to natural products than Gram negative bacteria [47
]. Indeed, Gram negative bacteria show more resistance to natural compounds since the hydrophilic cell wall structure of these bacteria is constituted of a lipopolysaccharide that blocks the penetration of hydrophobic compounds and other extracts in the target cell membrane.
From the PCA described above, it was possible to observe a correlation between some bacterial strains and some microalgal extracts. In the loading plot (Figure 5
B), a strong correlation was observed for Shewanella
sp. PVV6, Shewanella
sp. TC11, Pseudoalteromonas lipolytica
sp. 4M6 and Pseudoalteromonas
sp. 5M6 and most of the dinoflagellate extracts on the first axis whereas Shewanella
sp. MVV1 and Bacillus
sp. 4J6 allowed the discrimination of the other active extracts along the second axis (positively for C-59 and negatively for P-89). Noteworthy is the fact that anti-bioadhesion activities of most of the microalgal extracts against Shewanella
sp. MVV1 and Bacillus
sp. 4J6 were inversely correlated. Thus, three bacterial groups could be distinguished.
From these results, three bacterial strains and three microalgal extracts were selected for further investigations. Shewanella
sp. MVV1, Bacillus
sp. 4J6 and Paracoccus
sp. 4M6 were representative of the three bacterial groups. As shown previously in Table 2
, three extracts (P-43, P-60 and P-78) were the most active against the three selected bacteria. P-78 was active against the three strains whereas P-60 showed a remarkable inhibition of the adhesion of Bacillus
sp. 4J6 (86%) and P-43 of Shewanella
sp. MVV1 (82%).
Then, the concentration needed to inhibit 50% of the bacterial adhesion was determined for each microalgal extract. These EC50
values (Table 4
) ranged between 21 and 73 μg mL−1
. These results were on the same order of magnitude as those obtained by Camps et al. [34
] wherein EC50
values for commercial biocides against TC5, TC8 and 4M6 varied from 0.25 μg mL−1
to more than 160 μg mL−1
. As described above, the three extracts have shown a high anti-adhesion activity against the three bacterial strains. However, their impacts were variable depending on the strain. Bacillus
sp. 4J6 was significantly more sensitive to P-60 extract (p
< 0.05, Wilcoxon), whereas Paracoccus
sp. 4M6 and Shewanella
sp. MVV1 were significantly more affected by P-78 extract (p
< 0.05, Wilcoxon). Paracoccus
sp. 4M6 seemed to be the most resistant strain.
These results confirmed that the anti-bioadhesion activity of these selected microalgal extracts relied on a mechanism rather than a toxic effect and underlined the potentiality of these natural ingredients as antifouling agents.