Candida albicans
The C. albicans clinical isolate 50vr, used in the present study, was previously characterized as biofilm producer and highly virulent, as assessed by an in vivo infection model in Galleria mellonella [17]. C. albicans was kept in stock at − 20 °C and maintained for experiments by bi-weekly passages on Sabouraud Dextrose Agar plates. Fresh cultures were set the day before each experiment.
HSV-1
The HSV-1 strain used in this work was a clinical isolate, identified by monoclonal antibodies, laboratory adapted through serial passages (> 50) on VERO cells [14, 15]. The virus inocula employed in the experiments consisted of cell-free virus suspensions, obtained from centrifuged lysates of virus-infected VERO cells. Virus batches were titrated on VERO cells (108 PFU/mL) and kept frozen in aliquots at − 80 °C.
Cell line
The VERO cell line was used for the all experiments. Cells were cultured at 37 °C and 5% CO2 in minimum essential medium (MEM) with 10% (growth medium) or 5% (maintenance medium) foetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 μg/mL), ciprofloxacin (100 μg/mL) and l-glutamine (2 mM). The cell line was maintained by passages in fresh medium twice a week.
Laser source
The Laser Alba 355 (Elettronica Valseriana, Casnigo, BG, Italy) was used as UVA1 laser source; it works at 355 λ, allowing to set different programs by combining parameters such as laser power, time of exposition, distance from the laser source and shape of the radiated area.
Antiviral drugs
Two antiviral molecules were assessed against HSV-1, acyclovir (Recordati SpA, Milano, Italy) and foscarnet (Clinigen, Burton-on-Trent, UK). Both were commercial products commonly used for intravenous treatment.
Biofilm formation and exposure to HSV-1
Candida cells were grown overnight at 37 °C in yeast peptone dextrose (YPD), then harvested and washed with phosphate-buffered saline (PBS). After resuspension to 1 × 106 yeast cells/mL in MEM-10% FBS, 100 µL were seeded in duplicate in polystyrene, flat-bottom 96-well cell culture plates (Euroclone S.p.A., Pero-Mi, Italy) and incubated at 37 °C to allow biofilm formation, according to reported studies [14, 18, 19]. Twenty-four hours later, virus inoculum (50 μL, 107 PFU/mL final concentration) was added to biofilm-containing wells and to empty control samples. The samples were incubated for additional 24 h and then exposed to either physical or pharmacological treatments. Finally, the wells were scraped for 1′ with a plastic tip and the load of infectious virus embedded in the detached/rescued biofilm was titrated (see below). Each experiment was repeated 3–4 times and each condition was tested in triplicate.
In a further set of experiments aimed at assessing whether drug antiviral efficacy may be different when Candida is cultivated as biofilm or as planktonic, Candida was also seeded in wells with a glass cover slide on the bottom. In our hands, glass inhibits the Candida strain we used (50vr) in biofilm formation: in fact, Candida 50vr when cultured on glass surfaces grow in a planktonic pattern (personal observation) with an hyphal mass floating in the culture medium.
Virus titration
In each experiment, HSV-1 residual titer was established by end-point titration. At the end of each experiment, plate well content was harvested by scraping for 1′ with a plastic tip. After centrifugation, the rescued material was diluted with maintenance medium on a tenfold basis and each dilution was seeded in duplicate onto 24 h-old VERO cell cultures. After a 3 day incubation at 37 °C, the virus titer of each sample was established as the highest dilution showing the typical viral cytopathic effect. The results, expressed as tissue culture infectious dose 50 (TCID50)/mL, were calculated using the Reed and Muench formula [20].
In order to determine the Inhibitory Concentration 90 (IC90) or 50 (IC50) of the different treatments, the plaque reduction assay was used and it was performed according to published procedures [21]. After centrifugation of the rescued material, VERO cell monolayers were infected with tenfold serial dilutions of such material. After 1 h of incubation at 37 °C, virus inoculum was removed and each well was added with maintenance medium containing human γ-globulin anti-HSV-1 at 0.6% to neutralize non-penetrated virus. Medium was removed 2 days later, and the infected cell monolayers were fixed with methanol and stained with crystal violet (CV) to count the cytolysis plaques: in this case, virus titer was expressed as plaque forming units (PFU/mL).
Laser treatment of C. albicans
The effects of UVA1 were evaluated on C. albicans to determine whether the laser energy could have an inhibitory activity on biofilm. In particular, to test the effects of the laser beam on biofilm formation, Candida cells were exposed to the laser beam immediately after seeding the yeast cells in culture medium (pre-treatment) and then plates were incubated for 48 h to allow biofilm formation. Also, the effects of laser irradiation were evaluated on biofilm maintenance, by exposing mature biofilm to laser beam, namely 48 h after cell seeding (post-treatment). Nine different emission protocols, with energy ranging between 20 and 250 J/cm2, were applied. In any protocol tested, the laser beam had a square application and the tissue culture plate was set at 30 cm from the laser beam source. The metabolic activity and the total biomass of C. albicans treated and untreated biofilm were quantified by XTT assay and CV staining assay, respectively.
Crystal violet staining assay
CV staining assay was used to quantify the total biomass of control and laser-exposed C. albicans biofilms [22]. Briefly, wells containing Candida biofilm or controls (medium only) were washed 3 times with 200 µL of PBS and then air dried for 5′. After fixation with 100 µL of methanol for 20′, the samples were stained with 100 µL of 1% CV solution for 5′. Afterwards, each well was washed 3 times with 200 µL of distilled water and added with 33% acetic acid (100 µL/well). After 10 min, the optical densities (OD) were measured at 540 nm by a microplate reader (Sunrise, Tecan Group Ltd, Männedorf, Switzerland).
XTT assay
The XTT colorimetric tetrazolium assay was used to evaluate the effects of laser treatments on total metabolic activity of C. albicans biofilm [23, 24]. A commercial kit (AppliChem GmbH, Darmstadt, Germany) was employed following the manufacturer’s instructions. Briefly, wells containing Candida biofilm or control wells (medium only), exposed or not to the laser treatments (as above described), were washed 3 times with 200 µL of PBS and then 100 µL of the colorimetric solution were added. After 2 h incubation in darkness at 37 °C, the absorbance of the colored reduction product was measured by a spectrophotometer (Sunrise, Tecan Group Ltd, Männedorf, Switzerland) at an OD of 450 nm.
Assessment of virus sensitivity to laser treatment in the presence or absence of Candida biofilm
On the basis of XTT and CV assays results (see below), two laser protocols were employed: program A (sub-inhibiting treatment on Candida) which dispensed 50 J/cm2 and program B (treatment associated with a modest cytotoxicity on Candida) which dispenses 100 J/cm2. After 24 h incubation at 37 °C with virus, Candida biofilms were exposed or not to laser beam. Afterwards, washing and scraping for 1′ were performed and the load of infectious virus in the detached biofilm was determined by end-point titration on VERO cells. The same treatment was carried out also in culture wells without C. albicans (controls). The IC90 of UVA1 treatment on HSV-1 was determined with or without Candida biofilm. Thus, after 24 h of incubation at 37 °C and 5% CO2, biofilm and control cultures exposed to HSV-1 were treated with increasing laser fluencies from 10 to 60 J/cm2. Afterwards, the amount of residual HSV-1 in supernatants of detached materials was assessed by plaque reduction assay as detailed above.
Assessment of virus sensitivity to antiviral drugs in the presence or absence of Candida biofilm
Twenty-four hours old biofilms were exposed to VERO cells (2 × 105 cells/well) infected 24 h earlier with HSV-1 (multiplicity of infection: 30 PFU/cell); the co-cultures were performed in growth medium in the absence or presence of 50 µM acyclovir or 600 µM foscarnet. In parallel wells (with no Candida biofilm), HSV-1 infected VERO cells were treated with the same doses of the two antivirals. After 24 h of incubation at 37 °C, plates were frozen and thawed, then the viral titer of each well was determined by end-point titration on VERO cells.
In another set of experiments aimed at determining drug IC50, twofold dilutions of each drug were added to wells with infected VERO cells in the presence and in the absence of Candida biofilm. The viral yield in each group was titrated 24 h later by plaque assay. For acyclovir, concentrations ranging from 3 to 50 µM were assayed, while for foscarnet from 0.019 to 2.4 mM. A dose–response curve was then elaborated and the IC50 was determined for each drug, with or without Candida biofilm.
Statistical analysis
The data reported in figures are the mean values (± standard deviation) from at least three different experiments performed. The results were analyzed by the two-tailed Student’s t test and were considered significant when p < 0.05.