A novel study on the inhibitory effect of marine macroalgal extracts on hyphal growth and biofilm formation of candidemia isolates

Biofilm formation and hyphal growth are considered to be the most serious virulence factors of Candida species in blood causing candidemia infections, which are difficult to treat due to the spread of resistant Candida isolates to most antifungal drugs. Therefore, in this study, we investigated the effect of different types and concentrations of selected macroalgal extracts from Cladostephus spongiosus (Phaeophyta), Laurencia papillosa (Rhodophyta), and Codium arabicum (Chlorophyta) in inhibiting those virulence factors of the isolated Candida. Acetone extract of C. spongiosus (AECS) showed a stronger anticandidal activity against the selected strains than ethanol extract. Candida krusei was the highest biofilm producer among the selected isolates. AECS showed an inhibition of C. krusei biofilm formation as well as a reduction in the viability of preformed biofilms. Also, AECS reduced various sugars in the candidal exo-polysaccaride layer (EPS). Scanning electron microscopy (SEM) and light microscopic images revealed an absence of hyphae and an alteration in the morphology of biofilm cells when treated with AECS. Moreover, AECS downregulated the expression of hyphal specific genes, hyphal wall protein 1 (HWP1), Agglutinin-like protein 1 (ALS1) and fourth secreted aspartyl proteinase (SAP4), which confirmed the inhibitory effect of AECS on hyphal growth and biofilm formation. Gas chromatography-mass spectrophotometer (GC-MS) analysis of AECS showed three major compounds, which were non-existent in the ethanol extract, and might be responsible for the anticandidal activity; these revealed compounds were 4-hydroxy-4-methyl-2-pentanone, n-hexadecenoic acid, and phenol, 2-methoxy-4-(2-propenyl). These active compounds of AECS may be promising for future pharmaceutical applications in the treatment of candidemia.


Results
Algal extracts showed antifungal activity against the selected Candida species. Among different species of algae collected from the Red sea along the coastal region of Hurghada in Egypt, three species, namely, Cladostephus spongiosus, Laurencia papillosa, and Codium arabicum, were evaluated for their potential anticandidal activities. Table 1 revealed that C. spongiosus and L. papillosa extracts prepared with acetone and ethanol had active compounds that could inhibit growth of the four pathogenic Candida selected strains. While, the methanol extract of these algal extracts did not record an anticandidal activity against all the tested Candida. Acetone extract of C. arabicum showed the lowest anticandidal activity against the selected strains. However, the ethanolic and methanolic fractions of C. arabicum did not show any noticeable activity against all organisms. Further from the results obtained, it was observed that all the algal extracts prepared with acetone and ethanol could record higher inhibitory activities against the tested Candida compared to fluconazole, which did not show inhibitory activity at the same concentration of extracts (10 µg/ml). Among the algal extracts tested for inhibitory activities, acetone extract of C. spongiosus (AECS) showed relatively higher inhibitory activities (20.5, 18.0, 16.7 and 14.7 mm) against the selected Candida species (C. krusei, C. glabrata, C. parapsilosis, and C. albicans, respectively).
The MIC and MFC values with fungicidal ratios of AECS and fluconazole were shown in Table 2. Both MIC and MFC of AECS gave the lowest value of 80 and 320 µg/ml with a fungicidal ratio of 1:4 in C. krusei compared Algal extract ( www.nature.com/scientificreports www.nature.com/scientificreports/ to fluconazole which gave a fungicidal effect at very high concentration of 2000 µg/ml. C. krusei was the most susceptible strain to AECS treatment and interestingly, C. krusei was found to be the most prolific biofilm producing Candida strain (Fig. S2). Herein, we focus on the antifungal activity of AECS on C. krusei biofilm production and hyphal growth.

AECS inhibits biofilms formation and eradicates the performed biofilm. Activity of AECS on C.
krusei biofilm formation was quantified and viability was expressed in terms of metabolic activity percentage. The Biofilm inhibitory concentration (BIC) of AECS against C. krusei was 120 µg/ml (Fig. 1A). The BIC 80 (biofilm inhibiting concentration) was defined as the lowest concentration of AECS that inhibits 80% metabolic activity of biofilm formation as compared to control. Also, BEC 80 for C. krusei was 2-fold higher (240 μg/ml) compared to (BIC 80 = 120 μg/ml) (Fig. 1B), as BEC 80 (biofilm-eradicating concentration) was defined as the lowest concentration of AECS that eradicates 80% of performed biofilm compared to control.
SEM visualization of C. krusei biofilms. SEM observations provided useful information on the different cellular morphologies present in the biofilm structure. The effect of AECS on C. krusei biofilm and its cellular morphology was monitored by SEM (Fig. 2). SEM images of control plates showed the presence of dense complex structure of biofilm having hyphae and yeast cells ( Fig. 2A). In the presence of 80 μg/ml AECS, formation of biofilms was reduced with complete hyphal disappearance, and consisted mostly of yeast cells (Fig. 2B). At BIC 80 of AECS (120 μg/ml), biofilm cells were found to have perforated outer membrane with distorted shape (Fig. 2C). Few yeast cells with wrinkled surface can be seen at 160 μg/ml and 240 μg/ml of AECS concentration respectively (Fig. 2D,E). Further increase in AECS concentration (120 μg/ml) led to a complete inhibition of biofilms.

AECS inhibits the EPS production and hyphal growth of C. krusei. The major virulence factors of
Candida species include yeast-to-hyphal transition, and EPS production. EPS ensures the mechanical stability and the physical architecture of the formed biofilms. As a result, we used different concentrations of AECS (20, 40, and 80 µg/ml) to study their effects on the EPS production of C. krusei. Our results showed that the different tested concentrations of AECS reduced the amount of sugar content in treated C. krusei compared to the control as in Fig. 3 that showed the ability of AECS to decrease sugar content formed by C. krusei biofilms. Furthermore, our results showed that AECS inhibits the hyphal growth of C. krusei in a dose dependent manner (Fig. 4).
Microscopically, massive C. krusei hyphae were observed in control plates. In the meantime, hyphal growth was moderate at 20 μg/ml, and absent at 40 μg/ml of extract, indicating a directly proportional relation between concentration of AECS and inhibition of hyphal growth.

AECS downregulates C. krusei hyphal specific genes.
To determine possible molecular mechanism of AECS inhibition of C. krusei hyphal growth, we tested the expression level of hyphal growth associated genes such as HWP1, ALS1, and SAP4 genes. Expression of these genes in AECS treated cells was significantly reduced by 5-fold, 2.5-fold, and 3.3-fold, respectively, when compared to the control (Fig. 5).   www.nature.com/scientificreports www.nature.com/scientificreports/ Chemical analysis of the different C. spongiosus extracts using GC-MS. As a next step, it was necessary to check the chemical composition of the different C. spongiosus extracts using GC-MS. The chemical constituents, molecular weight and peak area of each component were listed in Tables (3, 4). Our results indicated that the major compounds in AECS were 4-hydroxy-4-methyl-2-pentanone (50.47%), n-hexadecanoic acid (6.46%) and Phenol, 2-methoxy-4-(2-propenyl) (9.21%). While, the three major compounds in the ethanol extract of C. spongiosus were 9,12,15-octadecatrienoic acid ethyl ester (22%), Hexadecanoic acid ethyl ester (16.4%), and 2-Hexadecen-1-ol,3,7,11,15-tetramethyl (15.25%). Preliminary screening suggested that these different major compounds in AECS might be the active compounds that cause C. krusei biofilm inhibition and hyphal growth. Meanwhile AECS is a mixture of several compounds, each component might contribute to the biofilm inhibition than if they acted alone. So, further study will be done for the isolation and the purification of active compounds with a comprehensive toxicological analysis to determine its safety as it is beyond the scope of this paper.

Discussion
Secondary metabolites of marine algae of potential interest have been extensively documented 25 . According to several reports, antimicrobial activity depends on algal species, extraction method, type of solvent and the resistance of the tested organism 26 . In the present study, acetone was the most effective solvent for the extraction of the bioactive compounds followed by ethanol. Furthermore, C. spongiosus was the most effective marine algae against the selected Candida species. These results are in agreement with many earlier reports 17,18 . Our data elucidated that AECS showed MIC and MFC at 80 μg /ml and 320 μg /ml against C. krusei. These results are consistent with the previous findings of Mickymaray and Allturaiki 28 who reported that U. prolifera demonstrated an MIC and MFC at 500 and 1000 μg/ml against A. niger.
Bioactive molecules of marine algal origin have high potentiality to subjugate the growth of many infectious organisms and to suppress their biofilm metabolic activity 26 . Biofilm formation and hyphal morphogenesis are   www.nature.com/scientificreports www.nature.com/scientificreports/ considered the most important virulence factors of Candida species 29 . The present study showed that secondary metabolites of AECS have the potential to attenuate these virulence factors.
AECS reduced the metabolic activity of the matured C. krusei biofilms in vitro and acts as a dominant antibiofilm agent that prevents biofilm formation and removes the existing biofilm. These results agreed with Dulger 30 who reported that AECS has antibacterial and antibiofilm activities. SEM images of the C. krusei biofilm demonstrated the presence of dense hyphae in absence of the extract. However, it showed deformed and swollen cells at BIC 80 . These morphological alterations of the cells resulting in cell death as reported previously for sophorolipid treatment against C. albicans 31 . Moreover, cells deformation and distortion of cell membrane have been reported as the mechanisms of antimicrobial activity for many biosurfactants 32 .
AECS suppressed the expression of hyphal genes illustrating the molecular mechanism of AECS in inhibition the hyphal growth. This result is in accordance with Haque 31 who reported the inhibition of C. albicans hyphal growth by sophorolipid using the same genes. To the best of our knowledge, there was not any scientific reports revealing the role of AECS against biofilm formation and hyphal growth of Candida sp.
As a next step, it was necessary to check the chemical composition of bioactive secondary metabolites in the different solvent extracts. The differences in the anticandidal effects of the algal extracts may be attributable to differences in the active compounds that present in the algae after their extraction with different solvents. The GC-MS analysis indicated that the chemical composition of the most promising AECS had 3 major peaks in comparison with the ethanolic extract; 4-hydroxy-4-methyl-2-pentanone was the major component, which showed the highest peak area percentage compared with the other components. On the other hand, this compound was not observed in the ethanolic extracts of C. spongiosus. Additionally, this compound had previously detected by GC-MS in acetone extract of the red algae Peterocladia Capillaceae and Laurencia pinnatifida showing a potent antimicrobial activity 33 . As well, this compound was detected as a volatile oil fraction from Phaeophyceae and Rhodophyceae that had an antimicrobial activity 34 . The second major component in AECS was n-hexadecanoic acid, which also was detected using GC-MS from Rhodophyceae 35 , and was reported to have an anticandidal activity 36 . In addition, n-hexadecanoic acid was found as a major component in the acetone extract of Sargassum hystrix with a strong antimicrobial activity 33 . Moreover, the third major component observed in AECS was Phenol, 2-methoxy-4-(2-propenyl), which previously identified by GC-MS analysis in methanol extract of Ulva lactuca with reported high antimicrobial and antioxidant activities 37 . Collectively, these results suggest that AECS is a mixture of several compounds, and each component might contribute to the biofilm inhibition than if they acted alone. Therefore, the current study suggested that the AECS is a potential source of natural anticandidal agents. It possessed certain metabolites with potent anticandidal properties that may be used for the treatment of blood candidemia infections as it can inhibit the candidal growth by suppressing biofilm formation, hyphal growth and its adhesion genes. Further study is required to characterize the antibiofilm activity of AECS in vivo by studying the antagonistic effect of its purified components against C. krusei biofilm and its safety.

Materials and methods
Organisms and growth conditions. Four Candida spp. strains (C. krusei, C. glabrata, C. parapsilosis, and C. albicans) were kindly provided by Dr. Mona Osama (Clinical Microbiology Unit, Tanta University Hospital, Faculty of Medicine, Tanta, Egypt). The selected strains were isolated from blood samples collected from the intensive care unit (ICU) and dialysis units in the Tanta University hospital in July 2016. All patients provided written informed consent and the study protocol was approved by the review board of Tanta University Hospitals for the collection of swabs from the Laboratories of Clinical Microbiology Unit at Tanta University Hospital, Faculty of Medicine, Tanta, Egypt. The clinicians followed the guidelines of the Declaration of Helsinki. One strain per patient was studied. Strains were stored at −70 °C. Phenotypic identification was confirmed with the API Candida system (bioMérieux Vitek, Hazelwood, MO, USA) following the manual instructions according to standard method of Buchaille 38 . The specific number code for each species is shown in the supplementary data (Fig. S1). A frozen glycerol stock of each strain was cultured on sabouraud dextrose broth (SDB; Ifco Laboratories, Detroit, MI, USA) and incubated at 37 °C for 24 h. Extraction of algal bioactive compounds using organic solvents. About 2 kg of the three isolated algal species were harvested, separately rinsed with sterile-filtered seawater and shade-dried, cut into small pieces, and powdered in a mixer grinder. Then, 5 g of powdered sample of each algal species was extracted separately and soaked with 40 ml of different solvents (acetone, ethanol and methanol) for 48 h. The obtained extracts were filtrated and concentrated in a rotatory evaporator at 40 °C. The residual solvent was removed with a vacuum pump. Then, the weighted crude extracts were well preserved in airtight containers and kept at −20 °C for further analysis 42,43 .

Anticandidal activity of selected algal extracts. An agar well diffusion method as detailed in
El-Zawawy and Hafez 44 was conducted to determine the most effective algal extract against the four selected strains. Briefly, sabouraud dextrose agar (SDA) plates were inoculated with 100 μl of each Candida strain (1 × 10 6 cells/ml) with wells of size 8 mm filled with 10 µg/ml of each algal extract dissolved in different solvents (acetone, ethanol and methanol). Each solvent (100 µg/ml) was added as a control, which did not show any antifungal activities (data not shown). Fluconazole (10 µg/ml) (Diflucan, Pizer) was used as a positive control. Then, these plates were incubated at 37 °C for 48 h. After the incubation period, the results were observed and the diameter of the inhibition zone around each well was measured to determine the most effective extract. All tests were performed in triplicate.

Determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC).
Minimal inhibitory concentration (MIC) of the most effective extract and fluconazole against the four selected strains was performed using 96-well microtiter plates. Selected strains were added in SDB supplemented with varying concentrations of acetone extract of C. spongiosus (AECS) and fluconazole, then incubated at 37 °C for 48 h. After incubation, the fungal growth was assayed at 600 nm using a Biotek plate reader. The MIC was recorded as the lowest concentration that produced complete suppression of visible growth 45 .
The MFC of AECS and fluconazole was determined according to Borman 45 . Briefly, (10 µg/ml) from MIC to last concentration wells of AECS and fluconazole were transferred separately to SDA plates, which were then incubated at 37 °C for 48 h. The MFC was recorded as the lowest drug concentration at which fungal growth was completely inhibited after 48 h of incubation.
Determining the cell viability of preformed biofilms in Candida strains. The ability to obtain quantitatively the metabolic activity of cells in preformed biofilms of the four Candida strains were tested by a reduction assay 46 using colorimetric XTT [2,3-bis (2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt]. A cell suspension of each Candida strain was prepared in SDB at a density of 1 × 10 6 cells/ml, after that 100 μl were added to each well in microtiter plates. The plates were incubated at 37 °C for 48 h. At the end of incubation, medium was aspirated from the wells and nonadherent cells were removed by washing the biofilms 3-times with a sterile phosphate buffered saline (PBS). Residual PBS of the wells was removed. To each well of prewashed biofilms, 900 μl of fresh broth, 90 μl of XTT salt solution (0.5 mg/ml) and 10 μl menadione solution (1 mM) were added and incubated at dark at 37 °C for 5 h. During incubation, biofilm metabolism reduces XTT tetrazolium salt to XTT formazan. Then, the absorbance was measured spectrophotometrically at 490 nm to obtain the strain which is the higher biofilm producer.
Effect of AECS on biofilm formation and preformed biofilm of the higher biofilm producer strain. The inhibitory activity of AECS on biofilm formation was assessed in vitro according to Ramage 47 . A cell suspension of the selected strain was prepared in SDB (1 × 10 6 cells/ml) and added to microtiter plates (100 μl per well) with 100 μl of different concentrations of AECS (10,20,40,80, 120, 180, 240 µg/ml). Similarly, 100 μl of SDB with 100 μl of acetone without algal extract were added into wells as a control. Microtiter plates were incubated at 37 °C for 48 h.

Quantification of exopolysaccharides (EPS) of AECS treated biofilm.
This assay was used to estimate the amount of exopolysaccharides in AEC treated preformed biofilm compared to untreated biofilm as a control. Preformed biofilms were prepared as described previously. The non-adherent cells were discarded and 500 µl of 0.9% NaCl was added to the wells of the plate and washed thoroughly. Then, cell suspensions in 0.9% NaCl were transferred to sterile test tubes with an equal volume of 5% phenol. Then, 5% v/v of concentrated sulfuric acid containing 0.2% hydrazine sulfate was added and incubated in dark for 1 h and the absorbance was measured at 490 nm according to Nithya 49 . Effect of AECS on candidal hyphal growth. Hyphal growth assay was performed in 10 ml of modified sabouraud glucose broth (MSGB) (Sigma) supplemented with 10% fetal bovine serum (FBS, Invitrogen). A cell suspension of the selected strain (1 × 10 6 cells/ml) was incubated with different concentrations of AECS (0, 20, 40, 80 μg/ml) at 37 °C with agitation (200 rpm) for 5 h. Aliquots of samples were stained using Lactophenol cotton blue and allowed to dry for 5 min, then visualized under a light microscope using an 40x objective lens and photographed (Nikon Eclipse Ti 100, Japan) 50 .

Gas chromatography-mass spectrometer (GC-MS) analysis. Different extracts from C. spongiosus
(acetone and ethanol) were investigated for their phytoconstituents using GC-MS (Trace GC Ultra, USA), at the National Research Centre (NRC), El Dokky, Giza Governorate. The identification of unknown compounds was based on comparing their retention time relative to those of the known compounds by matching spectral peaks available with Wiley 9 Mass Spectral Library 54 .
Statistical analysis. All data were expressed as mean ± standard deviation of three replicates and submitted to variance analysis using SPSS-20.

Data availability
The datasets used and analyzed during this study are available from the corresponding author upon request.