In this study, we demonstrated that corneal isolates of F. solani, C. sphaerospermum, and A. implicatum can form biofilms protected from antifungal drugs through time-dependent phases in vitro. To our knowledge, this is the first study to investigate the growth characteristics of C. sphaerospermum and A. implicatum biofilms, and to examine four distinct developmental phases of the biofilms in relation to antifungal susceptibility.
Our results demonstrated that
F. solani,
C. sphaerospermum, and
A. implicatum can produce biofilms through four basic time-dependent phases, including conidial adhesion, germling formation, microcolony formation, and biofilm maturation. It was consistent with those of other authors reporting on
Aspergillus biofilms.
13 It was observed that ECM was composed of galactomannan; α-1,3-glucans; monosaccharides; and polyols, melanin, and proteins.
18,19 In mature biofilms, crossing hyphae were glued by ECM, which was produced between the hyphae and surrounded them, and mature biofilms displayed water channel architecture that might represent an optimal arrangement for the influx of nutrients and disposal of waste products.
15 It was consistent with those of other authors reporting on
Fusarium biofilms formed on contact lenses.
10 Moreover, the growth patterns of biofilms formed by the fungi were different. We observed that
C. sphaerospermum took a long time to form a biofilm. Biofilms formed by
C. sphaerospermum were tightly attached to the surfaces of coverslips and not easily detached (data not shown), and its related mechanisms needed further investigation, which might therefore explain why the presentation of dematiaceous fungal keratitis represented a slowly progressive disease.
20 However, purposely designed experiments will be needed to further demonstrate this.
Fungal biofilms were more resistant to antimicrobial agents than planktonic cells. Four distinct growth phases in relation to antifungal susceptibility were examined. Our results demonstrated that all three strains became increasingly resistant to antifungal agents throughout morphological differentiation, which was consistent with the report by Imamura et al.,
10 showing that
Fusarium biofilms exhibited reduced susceptibility to lens care solutions in a time-dependent manner. Moreover, our results showed that the mature biofilms were intrinsically resistant to the azole antifungal drugs (FLU, VRC, and ITC). Multiple mechanisms have been proposed for the increased resistance of biofilms to antifungal agents. Our results indicate that ECM increased and a network of hyphal structures formed throughout the incubation time. The architecture of biofilms and the presence of ECM might reduce the diffusion of antifungal drugs, and they may be responsible for the increased resistance of biofilms to antifungal agents.
5,13 Moreover, differentially expressed genes might also increase resistance of biofilms to antifungal agents. Mukherjee et al.
17,21 demonstrated that efflux pumps had a critical role in FLU resistance in
Candida biofilms, and speculated that the resistance of
Fusarium biofilms to VRC might be due partly to upregulation of efflux pumps. Rajendran et al.
22 also reported that efflux pumps contributed to azole resistance in
Aspergillus biofilm; however, the genes contributing to the resistance of
F. solani,
C. sphaerospermum, and
A. implicatum biofilms to antifungal agents have yet to be determined, and purposely designed experiments will be needed to further demonstrate this point. Therefore, higher doses and early antifungal therapy should be considered for a better penetration of the drugs to the fungi.
The activity of antifungal drugs to different fungal biofilms was different. This study demonstrated that AMB had a wide spectrum of activity against various morphological forms of
F. solani biofilm, which was consistent with our previous report and the observations of Sengupta et al.
12,23 However, NAT was the most effective against 48-hour growth, which was consistent with the observations of Mukherjee et al.
17 Consistent with a previous report,
24 TRB was the most active agent against planktonic
A. implicatum cells; however, as in the development of biofilm, the activity of TRB was markedly reduced, and NAT was the most effective against 48-hour growth. Moreover, ITC was the most active agent against planktonic
C. sphaerospermum cells. As in the development of biofilm, the activity of ITC was reduced, and AMB was the most effective against mature biofilm. The ECM and hyphal structures of the three fungal biofilms were different from each other. The finding that fungal isolates varied in their ability to form biofilms and antifungal drugs exhibited varying activity against fungal biofilms was similar to a previous report. Mukherjee et al.
17 characterized the biofilms formed by
F. solani and
Fusarium oxysporum , and reported species-dependent antifungal susceptibilities of these two species. We presumed that different antifungal susceptibility of
F. solani,
C. sphaerospermum, and
A. implicatum biofilm might be due to the strains. Therefore, it was necessary to adjust treatment according to fungal strains and fungal biofilm growth phases.
In conclusion, F. solani, C. sphaerospermum, and A. implicatum can produce biofilms that were resistant to antifungal agents in vitro. Future studies are needed to demonstrate the potential impact of fungus biofilm on fungal keratitis.