The fungus and its maintenance
A. fumigatus ATCC46645, was obtained from the Culture Collection of the University of Westminster, London, UK. Stock cultures of A. fumigatus maintained on potato dextrose agar (PDA) (Merck, Dorset, UK,) medium were propagated in potato dextrose broth (PDB) (Fisher Scientific, Loughborough, UK) or RPMI-1640 (Merck, Dorset, UK).
Culture and treatment of A. fumigatus for resazurin-based viability assessment
Minimum inhibitory concentration (MIC) is defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation (Andrews 2001). Different doses of the antimicrobial agents, triclosan (Sigma-Aldrich, Dorset, UK) and L-AMB (Thermo Fisher Scientific, Leicestershire, UK) were applied to A. fumigatus at t0 and after 24 (represents early biofilm structure) and 48 (represents mature biofilm structure) hrs. To measure fungus viability, resazurin dye solution (Merck, Dorset, UK) was added to the culture (10% v/v) in 96-well plates. The plates were then incubated at 37°C on a shaker at 50 rpm for 50 min and the absorbance was measured spectrophotometrically at a wavelength of 590 nm. Single and combination treatments of A. fumigatus with antimicrobial agents, triclosan and L-AMB, were performed. For that, conidia and sessile (biofilm) cell viabilities were measured by In vitro toxicology assay kit (Merck, Dorset, UK) in 96-well tissue culture plates (Corning Inc., Corning, NY).
Treatment of A. fumigatus with triclosan and L-AMB
For single treatment, in a 96-well microplate, RPMI-1640 medium (200 µL) containing the agent’s effective dose and inoculum (106 conidia/mL) was added per well. After incubation at 37°C for 24 h, the cells’ metabolic activity was determined through resazurin assay.
Triclosan-L-AMB interactions were evaluated using the checkerboard assay. Checkerboards were prepared by using serial dilutions of L-AMB and triclosan. Triclosan and L-AMB dilutions were prepared as recommended in the EUCAST (European Committee on Antimicrobial Susceptibility Testing) protocol to give final drug concentrations of 0.3, 0.6 and 1.2 mg/L and 0.1, 0.2 and 0.4 mg/L for triclosan and L-AMB, respectively, in 100 µL of double-strength RPMI medium containing 106 conidia/mL fresh spore suspension. Subsequently, 100 µL of RPMI medium containing fresh spore suspension (106 conidia/mL) were added to the wells, resulting in concentrations of 0.15, 0.3 and 0.6 mg/L and 0.05, 0.1 and 0.2 mg/L for triclosan and L-AMB, respectively. For both simultaneous and continuous combination treatment strategies, viability was determined by using resazurin assay after 48 h treatment at 37°C. The OD at 570 nm wavelength was determined with a spectrophotometer (Jenway 6300 visible; Camlab Limited, UK). Fractional inhibitory concentration index (FICI) was calculated as follow:
(FICI=(MICAcomA,B÷MICagentA)+(MICBcomA,B÷MICagentB)) Equation 1
According to the above equation, FICI ≤ 0.5 indicates synergy, FICI > 4 indicates antagonism whereas 0.5 < FICI < 4 suggests no interaction. To study the synergistic assay, Compusyn software (ComboSyn, Inc.) was used. The resulting CI theorem of Chou-Talalay offers quantitative definition for additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1) in drug combinations (Chou 2010).
Evaluation of the EPS and biofilm depth by using confocal laser scanning microscopy
Leica SP2 LSCM (CLSM, Carl Zeiss, Jena, Germany) was used to examine the fluorescent filamentous biomass and hence the biofilm and its thickness. A. fumigatus biofilm formation on glass slides after 48 h of incubation at 37°C were analysed using FUN-1-based confocal laser scanning microscopy (CLSM). Microscopic visualization and image acquisition of biofilms were conducted using an upright scanning Leica confocal microscope equipped with an argon/krypton laser and detectors, and filter sets for monitoring of green (excitation 480 nm, emission 517 nm) and red (excitation 633 nm, emission 676 nm). The biofilms on the surfaces were washed three times in sterile PBS and stained using a fluorescent stain, FUN-1 (Molecular Probes, Life Tech) prepared according to the manufacturer’s instructions. For biofilm visualization, FUN-1 (1 µL) from a 10 mM stock was mixed in 1 mL of PBS. Three drops of the mixture were added on the top of the biofilm, which was then mounted on a coverslip. The slides were incubated for 45 min at 37°C in the dark. The biofilm, formed as explained above, was washed again with PBS and mounted on a slide. An excitation wavelength of 488 nm using an argon ion laser at a magnification of 200x was used to examine the biofilms. Horizontal (xy) view of reconstructed 3-dimensional images of FUN1-stained biofilms was applied to capture green fluorescence.
Thickness of the biofilm was observed in the side view of the reconstruction. Sections on the xy plane were taken at 1 µm intervals along the z-axis of the sections taken parallel to the x-y plane to determine the depth of the biofilms. The CLSM analysis of the biofilms was used to measure and to compare the “means” of the thickness of the triclosan-treated and the combination-treated test groups. Three-dimensional images were assembled using Leica Confocal Software.
Biofilm quantification by crystal violet assay
The effect of the agents (triclosan and L-AMB at their MICs) on the mature biofilm (36 h of incubation) was estimated using the crystal violet (CV) assay. The assay stains both live and dead cells as well as some components present in the biofilm matrix, thus it is well suited to quantify total biofilm biomass. To determine the ability to form biofilms, an inoculum (106 conidia/mL) was added to 200 µL PDB medium in a 96-well polystyrene microtiter plate, which was incubated, without agitation, at 37°C for 36 h. Subsequently, the medium was aspirated and non-adherent cells removed by thoroughly washing the formed biofilm three times with PBS. After fixation with glutaraldehyde (70% in H2O; Sigma-Aldrich, UK) for 20 min and air-drying, the biofilms were stained with 200 µL, 0.5% w/v CV (Merck, Dorset, UK) for 15 min, followed by rinsing with sterile PBS, and de-staining with 96% v/v ethanol (VWR, Brooklyn, NY). The absorbance of the CV- stained biofilm in the treated and untreated control groups was measured at 590 nm using a spectrophotometer. Percentage biofilm biomass in the treated samples was calculated using the following equation:
where A590 is the absorbance of the CV-stained biofilm matrix at 590 nm.
Viability assay of the conidia by using flow cytometry
propidium iodide (PI) is a positively charged, membrane-impermeable fluorochrome that can only pass through the membranes of stressed, injured, or dead cells. It has been already shown that flow cytometry can be applied on conidia cells to assay their viabilities (Balajee and Marr 2002; Mesquita et al. 2013; Vanhauteghem et al. 2017).
To assess conidia viability, protocol defined by Mesquita et al. 2013 was adopted and filtered sterile water was used instead of culture media. Flow cytometry tubes (3.5 mL) using control tubes and experimental tubes were prepared (1 mL of deionised water + 1 mL of the conidia suspension). Subsequently, 100 µL of each treated and untreated control group was added to a flow cytometry tube with 50 µL of PI (Abcam, Cambridge, UK) stock solution (1 mg/mL), and 1.85 mL deionised water. After 10 min of staining with PI, tubes were then mixed and analysed using the cytometer (NovoCyte Benchtop, ACEA Biosciences, UK). PI is excited at 488-523 nm. It fluoresces orange-red and can be detected using a 562-588 nm band pass filter. Conidial viability was defined by the FSC (forward scatter) and SSC (side scatter) characteristics and the mean fluorescence intensity (MFI) of PI in the treated and untreated conidia. Within scatter parameters, the pulse height vs pulse width plots (FSC vs SSC) are used to identify cells of interest based on size and granularity and to isolate single cells passing through the cytometer, thereby removing any non-single cells (doublets, clumps and debris) (Rowley 2012).
Real-time-PCR assay of ags3 and sph3 expressions
After 48 h static incubation, the fungal cultures were analysed for DNA quantification. Expression of the ags3 and sph3 was quantified by real-time polymerase chain reaction (RT- PCR) assay. RNA was extracted by using Allrep fungal DNA/RNA/protein kit. First strand synthesis was performed from total RNA with Quantitec Reverse Transcription kit (Qiagen, Manchester, UK). Forward and reverse primers (Eurofins Genomics, Germany) related to the genes of interest, ags3 and sph3 and a reference gene, sac7 were used (Table 1). A fragment of the gene encoding ags3 and sph3 was isolated from genomic DNA using PCR with master mix prepared by adding SYBR Green (Merck, Dorset, UK). RT-PCR was performed using a 7500 RT-PCR System (Fisher Scientific Ltd, Loughborough, UK). Fungal gene expression was normalized to A. fumigatus sac7 expression. A comparative threshold cycle (Ct) method was used for relative quantification detecting changes in expression of the genes of interest relative to a reference gene.
The SPSS software was used for paired sample T- Test calculation showing data sets that were deemed not significantly different (N.S. > 0.05) and data sets that were significant at different levels: *P ≤ 0.05, **P≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001 (SEM bars are shown for n=3).