Fungal cultivation and production of an antibacterial agent
A fungal isolate (ChemRU330) was used for the investigation. This fungus was obtained from Rhodes University, Grahamstown. Two 5 mm mycelial discs of the actively growing fungus was cultivated in a liquid modified Melin-Norkrans (MMN) medium and was incubated at 28 °C for three weeks, with continuous shaking at 150 rpm on a rotary shaker. After the incubation, the fermentation broth of the fungus was homogenised and filtered through Whatman no. 1 filter paper to obtain a cell-free crude filtrate.
DNA extraction
Genomic DNA was extracted from pure fungal mycelia using the ZR Fungal/Bacterial DNA Mini-Prep kit (Catalogue # D6005) according to manufacturer’s instructions. DNA concentration was determined by NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA) and preparations were diluted to make 1–5 ng/mL of DNA template. The integrity of the isolated DNA was evaluated by electrophoresis in a 1% (w/v) agarose gel at 100 V for 75 min in 1X Tris-borate-EDTA (TBE) buffer, stained with 2 μL (concentration 0.5 ng/mL) ethidium bromide and visualised under a Bio-Rad ChemiDoc X-Ray Spectrometer (XRS) system.
Amplification of the ITS and Cox1 gene regions
The method described by Nguyen and Seifert (2008) was used, where the ITS region was amplified using the following primers ITS1, ITS4 and ITS5 [11] and KAPA Taq ReadyMix (2X). The KAPA Taq ReadyMix (2X) is a ready-to-use cocktail containing all components for PCR, except primers and template. The 2X ReadyMix contains KAPA Taq DNA Polymerase (1 U per 50 μL reaction), KAPA Taq Buffer, dNTPs (0.2 mM of each dNTP at 1X), MgCl2 (1.5 mM at 1X) and stabilisers [12]. PCR master mix was made up in a total reaction volume of 50 μl comprising of 25 μL KAPA Taq ReadyMix PCR kit (KAPA Biosystems, Catalogue # KK1006), 5 μL template DNA, 2 μL each of both primers (forward and reverse), and 16 μL of water. Amplification was conducted in an automated Applied Biosystems 2720 Thermal Cycler. The cycling parameters used were as follows: initial denaturation at 95 °C for 3 min, 40 cycles at 95 °C for 45 s (denaturing), annealing temperature at 60 °C for 45 s, followed by extension at 72 °C for 1.5 min, and finally 72 °C for 8 min (final elongation). After that, electrophoresis was used as previously described to determine the size of the amplified bands. The Cox1 gene region was amplified using primers designed for the Cox1 gene of the Pezizomycotina, PezizF (5′-TCAGGRTTAYTAGGWACAGCATTT-3′) and PezizR (5′-ACCTCAGGRTGYCCGAA GAAT-3′) [3]. The PCR amplification was carried out in a total reaction volume of 25 μL comprising of 12.5 μL KAPA HiFi HotStart ReadyMix PCR kit (KAPA Biosystems, Catalogue # KK2605), 5 μL template DNA, 0.75 μL each of both primers (forward and reverse) and 6 μL of water. An automated Applied Biosystems 2720 Thermal Cycler was used. The initial denaturation temperature was set at 95 °C for 3 min, denaturation temperature at 95 °C for 1 min (40 cycles), annealing temperature 51 °C for 1 min, extension temperature 72 °C for 1 min 30 s, and final elongation temperature 72 °C for 8 min [3]. After that, electrophoresis was used as previously described to determine the size of the amplified bands.
Sequencing and phylogenetic analysis
After amplification of ITS and Cox1 barcode regions, the PCR products were cleaned up using a Wizard SV gel, and PCR clean-up kit (Promega, Catalogue # A9281) and the protocol outlined by the manufacturer of the kit was followed. The purified PCR products were sent to Inqaba Biotechnology, Pretoria, South Africa for Sanger sequencing. The sequencing reaction was carried out using the respective primers for IITS region and Cox1 gene [13, 14]. Nucleotide sequence chromatograms were analysed and edited using Chromas Lite software and compared to sequences in National Centre for Biotechnology Information (NCBI) http://www.ncbi.nlm.nih.gov [15] and UNITE https://unite.ut.ee [16] databases using Basic Local Alignment Search Tool (BLAST) program. Sequences derived from the study and their respective closest matches with homology greater than 95% were pre-aligned in Chromas version 2.6.4 (www.technelysium.com.au) before alignment using BioEdit sequence alignment editor version 6 [17]. To test for phylogenetic relationships, the ChemRU330 sequence and those of species in the genera Leohumicola (available in GenBank) were aligned using the ClustalX Version 1.81 [18]. Phylogenetic analysis of Cox1 barcode region was performed using molecular evolutionary genetics analysis version 7 (MEGA 7) [19]. The evolutionary history was inferred using Neighbor-Joining (NJ) statistical method [20]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [21]. Bootstrap support values above 50% from 1000 replicates search. The evolutionary distances were computed using Maximum Composite Likelihood method [22] and are in the units of the base number substitutions per site. The analysis involved 25 nucleotide sequences and codon positions included were 1st + 2nd + 3rd + noncoding. All positions containing gaps and missing data were eliminated. There were a total of 628 bp in the final dataset. Myxotrichum deflexum was chosen as an outgroup to analysis because of its status as near neighbour to Leohumicola clade in the 18S analyses [4].
Test bacteria
The crude extract of each fungal isolate was screened for antibacterial activity using some bacterial strains as indicator organisms. The indicator bacteria included both Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli, Serratia marcescens, Proteus vulgaris, Shigella sonnei and Klebsiella pneumoniae) bacteria, and were obtained from the undergraduate laboratory of the Department of Biochemistry and Microbiology, Rhodes University, Grahamstown. The bacterial isolates were already identified cultures used for undergraduate practical demonstration. All bacterial cultures were adjusted to 0.5 McFarland standards, which is visually comparable to a microbial suspension of approximately 1.5 × 108 CFU/mL.
Preliminary screening (using crude fungal filtrate)
The initial screening of antibacterial activity was conducted using a well-dilution method. The nutrient agar (NA) and Luria-Bertani (LB) media were poured into separate Petri plates and inoculated with 50 μL of the bacterial suspension (1.5 × 108 CFU/mL) and spread uniformly by using a sterile glass spreader. Wells (5 mm) were made on the agar media with a sterile cork borer, 50 μL crude filtrate of each of the fungal isolate was placed into each separate well, and the controls [positive (chloramphenicol) and negative (sterile distilled water)]. Parafilm was used to seal the plates before incubation at 37 °C for 24 h. Plates showing antibacterial activity were confirmed by visualisation, followed by measurement of inhibition zones. The average of three repeated trials was taken to evaluate the antibacterial activity [23].
Qualitative phytochemical screening
Phytochemical screening was conducted on the ethyl acetate extract to check for the presence of the following secondary metabolites – alkaloids, flavonoids, phenols, saponins, steroids, cardiac glycosides, tannins, and terpenoids. Alkaloids: an 80 mg of solid fungal extract was dissolved in 4 mL 2 N HCl. The sample was divided into two portions, one portion was treated with equal amount of Wagner’s reagent, and the second portion was treated with equal amount of Mayers reagent. Reactions showing the appearance of a brown precipitate indicated the presences of alkaloids. Flavonoids (zinc hydrochloride reduction test): to a test-tube containing 1 mL of fungal crude extract, 5–10 drops of dilute HCl, 0.5 g of zinc turnings were added, and the solution was boiled for 2 min. A reddish pink or dirty brown colouration of the solution indicated the presences of flavonoids in the extract. Phenols: a 40 mg crude extract was dissolved in 2 mL of distilled water. Then, a few drops of neutral 5% FeCl3 solution was added. A dark green colour indicated the presence of phenolic compounds. Saponins: The crude extract (1 mL) was combined with 5 mL water and shaken for 2 min. The saponins are known to possess frothing activity, the volume of foam was recorded every 10 min. Froth more than 1.5 cm indicated a positive result. Steroids: steroid content was detected by using the Liebermann-Burchard reaction method. A 1 mL fraction of the crude extract was placed in a tube containing acetic anhydride, and a few drops of sulphuric acid (H2SO4) was added. A bluish-green ring indicated the presence of steroids. Cardiac glycosides: a 1 mL FeCl3 reagent (a mixture of 1 volume of 5% FeCl3 solution and 99 volumes of glacial acetic acid) was added to 1 mL of the crude extract and was later treated with a few drops of H2SO4, carefully placed in a dropwise manner along the sides of the test tube. The appearance of greenish blue colour within a few min indicated a positive result. Tannins: the already prepared alcoholic FeCl3 reagent was mixed with the crude extract. The mixture produced a bluish-black colour, which disappears on the addition of a few drops of H2SO4 to yield a yellowish brown precipitate indicates a positive result. Terpenoids: a 1 mL crude extract was added to 1 mL of chloroform, followed by addition of 1 or 2 drops of concentrated H2SO4 to form a layer. A reddish-brown precipitate at the interface indicated that terpenoids were present [24, 25].
Extraction and concentration of crude antibacterial compound
The bioactive compound of the fungal filtrate showing inhibition after preliminary screening was then extracted by solvent extraction procedure using ethyl acetate as the organic solvent. To the filtrate, an equal volume of extracting solution (ethyl acetate) was added (ratio 1:1), this was mixed thoroughly for 10 min and kept for 5 min to obtained two clear immiscible layers. The upper tier (bioactive compound) was separated using a separating funnel. The extracting solvent was then evaporated, and the resultant compound was dried in a rotary vacuum evaporator (Buchi Rotavapor® R-200 Rotavapor System) to yield the crude metabolite [26]. The crude extract was then dissolved in distilled water and kept at 4 °C. A known weighed crude extract was dissolved in distilled water before use to obtain a particular concentration.
Determination of MIC and MBC (cell viability assay using MTT) by microbroth dilution method
The concentrated crude extract was adjusted to a final concentration of 16 mg/mL using a sterile distilled water as the diluent. The test was carried out in duplicate. The MIC was determined using the micro broth dilution method in a 96-well microtiter plate. The test organisms were grown for 24 h at 37 °C. 100 μL of bacterial liquid culture [(optical density adjusted to match 0.5 McFarland standard (1.5 × 108 CFU/mL)], then distributed into a 96-well microtiter plate. The crude extract preparations were diluted to contain different concentrations ranging from 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 and 0.03125 mg/mL dilutions, and were added to the wells containing the bacterial cells. A 5 μg/mL chloramphenicol was used as positive control, and sterile distilled water was used as negative control for the test microorganisms, and the microtiter plates were incubated at 37 °C for 24 h [27]. A 40 μl (0.2 mg/mL) thiazolyl blue tetrazolium bromide [methylthiazolyldiphenyl-tetrazolium bromide (MTT)] was added and at 37 °C for 30 min to detect the MIC. The presence of viable bacterial cells reduced the yellow dye to a pink colour. MIC is the lowest concentration that prevented change and inhibited bacterial growth. MBC was determined by removing a portion of liquid (50 μL) from each well without colour change and placing it on NA, and LB agar then incubated at 37 °C for 24 h. The lowest concentration that yielded no growth after this culturing was regarded as the MBC. All experiments were performed in duplicate [28].
