2.3. Activity against Biofilm Maturation
Extracts were then tested against the growth of C. albicans
biofilms on polystyrene substrates (anti-maturation activity) by incubating with very young biofilms (2 h adhered cells) for 24 h or 48 h. The half maximal inhibitory concentrations (IC50
) of the extracts are presented in Table 2
. Eleven extracts significantly inhibited biofilm growth onto polystyrene surfaces (1.5 µg/mL ≤ IC50_mat 48h
≤ 100 µg/mL; p
< 0.03) after a 48 h incubation, suggesting their ability to delay biofilm maturation.
Four extracts (Anaptychia ciliaris, Cetraria islandica, Peltigera rufescens, and Xanthoria parietina) were not promising as anti-maturation agents due to the lack of activity at 24 h and their low activity at 48 h and were not selected for further investigations.
Among the active extracts, only four (Evernia prunastri, Peltigera hymenina, Ramalina fastigiata, and Xanthoparmelia conspersa) were active after both 24 h and 48 h incubation time, but the IC50 obtained after 24 h were always higher (25 µg/mL ≤ IC50_mat 24h ≤ 100 µg/mL) than those observed after 48 h (1.5 µg/mL ≤ IC50_mat 48h ≤ 25 µg/mL). This observation suggests that an extended contact between extracts and microbes is needed to obtain overall inhibitory activity. Additionally, it also suggests that these extracts could be promising for long-term prophylactic action. It is noteworthy that E. prunastri and R. fastigiata were the most active extracts against maturation phase, with IC50_mat 24h = 25 µg/mL and IC50_mat 48h ≤ 3.1 µg/mL.
2.4. Anti-Biofilm Activity
Lichen extracts which demonstrated an anti-maturation activity with IC50_mat 48h
≤ 50 µg/mL have been selected and tested against a pre-formed C. albicans
biofilm 24 h of age by incubation for 48 h (according to the previous observation with anti-maturation tests), to evaluate their interest for a curative approach. Two complementary methods were used, tetrazolium salt (XTT) assay and colony-forming unit (CFU) counts. The IC50
and percentage of inhibition of the extracts are presented in Table 2
. The seven tested extracts demonstrated significant anti-biofilm activity (≤10 µg/mL ≤ IC50_biof
≤ 100 µg/mL; p
Four extracts, C. uncialis
, E. prunastri
, R. fastigiata
, and X. conspersa
showed the highest activity with IC50_biof
≤ 10 µg/mL. CFU counts confirmed this anti-biofilm activity by demonstrating a significant decrease in the yeast community constituting the biofilm (inhibition ≥ 80%; p
< 0.04) (Table 2
). This decrease was also confirmed by direct observations (inverted optical microscopy) (data not shown). This multi-method approach makes it possible to be sure that there is no interference between lichen extracts and XTT, leading to false positives.
Additionally, microscopic observations performed at the end of anti-biofilm tests using trypan blue staining showed that yeasts growing as biofilm were still alive after treatment (all cells were colorless), suggesting a non-lethal effect of the active extracts. Thus, these preliminary observations and results suggest that active extracts may act thanks to a dispersant or removing action that would not disrupt the fungal cell membrane.
The anti-biofilm activity of the studied lichens is poorly documented and no bibliographic data reported anti-biofilm activity for the tested extracts. Thus, results obtained for the seven lichens whose extracts have demonstrated anti-maturation and anti-biofilm activities are innovative in this field of research.
2.5. Comparison of Chemical Profiles of Active and Inactive Extracts
Regarding the compounds in active and inactive extracts, some metabolites did not appear to be involved in the anti-maturation effect observed. Thus, the dibenzofurans placodiolic, pannaric, or didymic acids (predominant in Leprocaulon microscopicum, Lepraria membranacea, and Cladonia incrassate, respectively), the depsides atranorin and thamnolic acid (predominant in Pseudevernia furfuracea and Cetrelia olivetorum for atranorin, and predominant in Cladonia parasitica, C. squamosal, and Usnea florida for thamnolic acid), the depsides with aliphatic chains divaricatic and perlatolic acids (present in Neofuscellia pulla); the tridepsides gyrophoric acid and tenuiorin (predominant in Lasallia pustulata and Peltigera species), the depsidone norstictic acid (predominant in Pleurosticta acetabulum) or the aliphatic acid caperatic acid (predominant in Flavoparmelia caperata and Platismatia glauca) and rocellic acid (Lepraria membranacea), and the triterpene zeorin (present in Leprocaulon microscopicum) were reported in inactive extracts. Lichens with a chemical profile dominated by terpenoids, (in the genus Peltigera), were also mostly inactive.
Some compounds (such as usnic, fumarprotocetraric, protocetraric, salazinic, and squamatic acids), were present both in active and inactive extracts. Their role in the biological activity is more questionable. Focusing on the widespread dibenzofuran, usnic acid, a weak activity has been shown for lichens containing high proportions of this metabolite. This observation was confirmed by a HPLC dosage using a calibration curve with usnic acid (Supplemental Figure S2
). Usnic acid represent 40% of the dried weight of the extract in Usnea florida
and 20% of the dried weigh of the extract for Flavoparmelia caperata
]. This result suggested the poor implication of this compound against the development and maturation of Candida
Finally, some compounds (stictic acid, evernic acid, parietin) were predominant only in active extracts (X. conspersa
, E. prunastri
, R. fastigiata
, and X. parietina
) suggesting their implication in the anti-maturation process of C. albicans
biofilms (Supplemental Figure S1
The extracts with the highest activity against both growing biofilms and 24 h old biofilms, E. prunastri
and R. fastigiata
, were dominated by the presence of the previously-mentioned evernic acid [29
]. E. prunastri
and evernic acid have already been studied for their antiproliferative and antimicrobial activities [31
]. In a recent publication, the effect of evernic acid on bacterial biofilm has been evaluated and this depside has demonstrated a capacity to inhibit Pseudomonas aeruginosa
quorum sensing systems [33
Stictic acid, is a depsidone dominated the chemical composition of the extract of X. conspersa
]. Antioxidant, antimicrobial, and apoptotic effect have already been described for stictic acid but no anti-biofilm activity is reported [35
]. We suggested here the possible interest of this compound against C. albicans
The other Xanthoparmelia, X. tinctina, has a complex chemical composition dominated by usnic acid and the depsidone salazinic acid. Surprisingly, salazinic acid is also present in lichens without any activity suggested possible synergistic mechanism inside this extract.
Another depside, squamatic acid, whose structure is closely related to evernic acid, is associated to usnic acid in C. uncialis
extract ,and could also be involved in anti-biofilm activities [26
]. The large proportion of (−)-usnic acid in this extract invites to distinguish the difference of activity between the two isomers. Studies reported activities of usnic acid against bacterial biofilms (Staphylococcus
, etc.) but also against fungal biofilms [37
]. A recent study demonstrated that usnic acid exhibited a significant biofilm inhibition against azole-resistant and azole-sensitive C. albicans
strains (71.08% and 87.84%, respectively). This effect would be mediated by oxidative and nitrosative stress, with a significant accumulation of intracellular and extracellular reactive oxygen species [41
]. Usnic acid also inhibited the yeast to hyphal switch and reduced the thickness of matured biofilms of C. albicans
]. Furthermore, it was able to reduce various sugars present in the exopolysaccharide layer. However, this compound does not appear to be effective against C. krusei
biofilms and the effect is controversial against C. parapsilosis
extract is constituted by a mixture of terpenoids and unidentified depsides and the role of each compound in the activity needs to be further established. The chemical profile of C. ramulosa
, showed the presence of fumarprotocetraric acid along with another unidentified compound (Supplemental Figure S1
Thus, this preliminary screening is a springboard towards additional investigations on lichen metabolites. Indeed, the pure compounds will be isolated in sufficient quantity from lichens and will be evaluated. If the activity against biofilms is not confirmed, synergistic effects and identification and isolation of minor compounds will be undertaken.