Antimicrobial Metabolites against Methicillin-Resistant Staphylococcus aureus from the Endophytic Fungus Neofusicoccum australe

Antimicrobial bioassay-guided fractionation of the endophytic fungi Neofusicoccum australe led to the isolation of a new unsymmetrical naphthoquinone dimer, neofusnaphthoquinone B (1), along with four known natural products (2–5). Structure elucidation was conducted by nuclear magnetic resonance (NMR) spectroscopic methods, and the antimicrobial activity of all the natural products was investigated, revealing 1 to be moderately active towards methicillin-resistant Staphylococcus aureus (MRSA) with a minimum inhibitory concentration (MIC) of 16 µg/mL.


Introduction
In 2014, the World Health Organization (WHO) described how drug-resistant microbes are present in every region of the world [1]. The report concluded that within a decade, antimicrobial resistance will make routine surgery, organ transplantation, and cancer treatment life-threateningly risky [1]. Key to managing this crisis is to boost the number of new antibiotic classes reaching the clinic [2,3]. The International Collection of Microorganisms from Plants (ICMP), curated by the Crown Research Institute Manaaki Whenua, has over 10,000 fungal cultures derived from plants and soil from Aotearoa New Zealand and the South Pacific. The collection has a great diversity of fungal species, host substrates, and collection localities, with the earliest cultures dating from the early 1960s [4].

Results and Discussion
Antimicrobial screening of ICMP isolates against antibiotic-sensitive and antibioticresistant strains of Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus identified Neofusicoccum australe as a hit (see supporting information). During the testing of fungal crude extracts against E. coli ATCC 25922 and S. aureus ATCC 29213, activity was observed primarily against S. aureus ( Figure 3). Initial fractionation of the crude extract was conducted by C8 reversed-phase column chromatography, eluting with a gradient of H2O/MeOH, to afford five fractions (F1-F5). Antimicrobial testing of F1-F5 against the same two microbes identified S. aureus activity in F3, F4 and F5 ( Figure 3). The most potent inhibitory activity was observed in F5, which displayed inhibition of S. aureus ATCC 29213 in a dose-dependent manner with a minimum inhibitory concentration (MIC) of 32 µg/mL. Further purification of combined F4 and F5 led to the isolation of compounds 1-4, while purification of F3 afforded compound 5. included two naphthalene monomers (2 and 3) [10], pramanicin A (4) [11] and 4-hydroxyscytalone (5) [8], was conducted by comparison of 1 H NMR data with those reported in the literature. Herein, the isolation, structure elucidation and bioactivity of neofusnaphthoquinone B (1) are described.

Results and Discussion
Antimicrobial screening of ICMP isolates against antibiotic-sensitive and antibioticresistant strains of Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus identified Neofusicoccum australe as a hit (see supporting information). During the testing of fungal crude extracts against E. coli ATCC 25922 and S. aureus ATCC 29213, activity was observed primarily against S. aureus ( Figure 3). Initial fractionation of the crude extract was conducted by C8 reversed-phase column chromatography, eluting with a gradient of H2O/MeOH, to afford five fractions (F1-F5). Antimicrobial testing of F1-F5 against the same two microbes identified S. aureus activity in F3, F4 and F5 ( Figure 3). The most potent inhibitory activity was observed in F5, which displayed inhibition of S. aureus ATCC 29213 in a dose-dependent manner with a minimum inhibitory concentration (MIC) of 32 µg/mL. Further purification of combined F4 and F5 led to the isolation of compounds 1-4, while purification of F3 afforded compound 5.

Results and Discussion
Antimicrobial screening of ICMP isolates against antibiotic-sensitive and antibioticresistant strains of Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus identified Neofusicoccum australe as a hit (see supporting information). During the testing of fungal crude extracts against E. coli ATCC 25922 and S. aureus ATCC 29213, activity was observed primarily against S. aureus ( Figure 3). Initial fractionation of the crude extract was conducted by C 8 reversed-phase column chromatography, eluting with a gradient of H 2 O/MeOH, to afford five fractions (F1-F5). Antimicrobial testing of F1-F5 against the same two microbes identified S. aureus activity in F3, F4 and F5 ( Figure 3). The most potent inhibitory activity was observed in F5, which displayed inhibition of S. aureus ATCC 29213 in a dose-dependent manner with a minimum inhibitory concentration (MIC) of 32 µg/mL. Further purification of combined F4 and F5 led to the isolation of compounds 1-4, while purification of F3 afforded compound 5.
The isolation of 1 as a racemic mixture is not uncommon for this class of natural products [8,9]. To the best of our knowledge, this is the fourth example of an unusual class of natural products which contain two naphthoquinone subunits connected in a head-to-tail fashion by an ethylidene linker [7][8][9]. The known compounds were identified as 6-(1-hydroxyethyl)-2,7-dimethoxyjugalone (2) [10], 6-(1-ethyl)-2,7-dimethoxyjugalone (3) [10], pramanicin A (4) [11] and (3S,4S)-4-hydroxyscytalone (5) [8].  The antimicrobial activity of 1-3 and 5 was evaluated against a panel of Gram-positive (methicillin-resistant S. aureus) and Gram-negative (Pseudomonas aeruginosa, E. coli, K. pneumoniae and Acinetobacter baumanii) bacteria and two fungal strains (Candida albicans and Cryptococcus neoformans) ( Table 2). The antimicrobial activity of pramanicin A (4) was not investigated in the present study due to lack of sample; however, the antifungal activity of the natural product has been previously reported [15]. Both neofusnaphthoquinone B (1) and monomer 2 exhibited activity against MRSA with 1 exhibiting more potent activity than 2. Interestingly, during initial ZOI screening, ICMP 21498 (see supporting information) appears equally potent against both S. aureus and E. coli isolates; however, this indiscriminate killing does not appear to have persisted past extraction. It is, therefore, a possibility that several compounds may be responsible for the antibacterial activity of ICMP 21498 and the other compound(s) were not extracted. Compound The antimicrobial activity of 1-3 and 5 was evaluated against a panel of Gram-positive (methicillin-resistant S. aureus) and Gram-negative (Pseudomonas aeruginosa, E. coli, K. pneumoniae and Acinetobacter baumanii) bacteria and two fungal strains (Candida albicans and Cryptococcus neoformans) ( Table 2). The antimicrobial activity of pramanicin A (4) was not investigated in the present study due to lack of sample; however, the antifungal activity of the natural product has been previously reported [15]. Both neofusnaphthoquinone B (1) and monomer 2 exhibited activity against MRSA with 1 exhibiting more potent activity than 2. Interestingly, during initial ZOI screening, ICMP 21498 (see supporting information) appears equally potent against both S. aureus and E. coli isolates; however, this indiscriminate killing does not appear to have persisted past extraction. It is, therefore, a possibility that several compounds may be responsible for the antibacterial activity of ICMP 21498 and the other compound(s) were not extracted. Both natural products 1 and 2 were also screened for cytotoxicity against human embryonic kidney cells (HEK293) and red blood cell haemolytic properties (Table 3). No cytotoxicity or haemolytic activity was observed for both 1 and 2, which, combined with the moderate antimicrobial activity against MRSA, makes these compounds of interest. All values presented as the mean (n = 2). a Concentration of compound at 50% cytotoxicity on HEK293 human embryonic kidney cells. Tamoxifen was the positive control (IC 50 9 µg/mL, 24 µM); b concentration of compound at 10% haemolytic activity on human red blood cells. Melittin was the positive control (HC 10 2.7 µg/mL); c not active at a single dose test of 32 µg/mL.

General Experimental Procedures
Melting points were measured on a Reichert melting point apparatus (Reichert, Vienna, Austria). Infrared spectra were recorded on a Perkin Elmer Spectrum 100 Fourier Transform infrared spectrometer (PerkinElmer, Boston, MA, USA) equipped with a universal ATR accessory. V max are expressed with units of cm −1 . Ultraviolet-visible spectra were acquired using a UV-2101 PC UV-Vis scanning Shimadzu spectrophotometer (Shimadzu, Kyoto, Japan) with a pair of 1 cm path length rectangular quartz cuvettes (3 mL, type 3) to measure λmax and log ε expressed in units of nm. NMR spectra were recorded using a Bruker Avance DRX-400 spectrometer or an Avance III-HD 500 spectrometer (Bruker, Karlsruhe, Germany) operating at 400 or 500 MHz for 1 H nuclei and 100 or 125 MHz for 13 C nuclei utilizing standard pulse sequences at 298 K. Chemical shifts are expressed in parts per million (ppm) relative to the residual non-deuterated solvent in 1 H-NMR and to deuterated solvent in 13 C-NMR (CD 3 OD: δ H 3.31, δ C 49.0; DMSO-d 6 : δ H 2.50, δ C 39.52). For 1 H-NMR, the data are quoted as position (δ), relative integral, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, dq = doublet of quartets, m = multiplet, br = broad), coupling constant (J, Hz) and assignment of the atom. The 13 C-NMR data are quoted as position (δ) and assignment of the atom. High resolution mass spectra were recorded on a Bruker micrOTOF QII (Bruker Daltonics, Bremen, Germany). Analytical thin layer chromatography (TLC) was carried out on 0.2 mm thick plates of DC-plastikfolien Kieselgel 60 F254 (Merck, Munich, Germany). Reversed-phase column chromatography was carried out on C 8 support with a pore size of 40-63 µm (Merck, Munich, Germany). Gel filtration chromatography was carried out on Sephadex LH-20 (Pharmacia). Flash chromatography was carried out on Diol-bonded silica with a pore Molecules 2021, 26, 1094 6 of 10 size of 40-63 microns (Merck, Munich, Germany). Analytical reversed-phase HPLC was run on a Dionex UltiMate 3000RS system (Waltham, MA, USA) using a C 8 column (3 µm Econosphere Rocket, 7 × 33 mm) (Grace, Columbia, MA, USA) and eluting with a linear gradient of H 2 O (0.05% TFA) to MeCN over 20 min at 2 mL/min. All solvents used were of analytical grade or better and/or purified according to standard procedures. Chemical reagents used were purchased from standard chemical suppliers and used as purchased.

Fungal Material
The ascomycete fungus Neofusicoccum australe (ICMP 21498) was isolated from diseased grapevines in New Zealand [16]. The isolate was identified based on a match of the sequence of the fungal barcode locus ITS (GenBank: MT107904) to reference specimens.

Antimicrobial Activity of Fungal Cultures
Pre-screening of the ICMP fungal cultures for antimicrobial activity involves briefly growing the cultures on potato dextrose agar (PDA) before small wells are cut into the agar and each well inoculated with 5 × 10 6 colony forming units of luciferase-tagged derivatives of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. The cultures are incubated, and the inhibitory activity of the ICMP isolates monitored by the extent of reduction in bacterial light production compared to bacteria isolated with no fungus.

Antimicrobial Testing of Extracts
Dry samples of extracts were dissolved in DMSO to make a 25 mg/mL solution and then further diluted into Mueller Hinton broth II (MHB) to achieve a maximum concentration of 2 mg/mL. Each extract (200 µL) was added to two adjacent wells along the top of the 96-well plate (Thermo Fisher, NUN167008, Waltham, MA, USA). MHB (100 µL) was then added to the remaining wells and extract solution (100 µL) serially diluted twofold down the plate and discarded. Aliquots of bacteria, S. aureus ATCC 29213 and E. coli ATCC 25922, at an optical density at 600 nm of 0.01 (approximately 1 × 10 6 colony forming units (CFU)/mL) were then added to all the wells. This gave a maximum concentration of 1 mg/mL and a minimum concentration of 16 µg/mL. The maximum volume/volume concentration of DMSO in all extracts was 4%; therefore, the negative control was tested at an identical concentration.
Absorbance was measured at 600 nm using an Enspire plate reader (Perkin Elmer, MA, USA) at 0, 2, 4 and 20 h to determine the minimum inhibitory concentration (MIC), between which times the plates were incubated at 37 • C with shaking at 100 rpm. After 20 h, 10 µL of liquid from all wells showing inhibition of bacterial growth was pipetted onto a plate of MH agar. Once all liquid had evaporated, the plates were then incubated inverted at 37 • C for 16-20 h, and the minimum bactericidal concentration (MBC) was measured [5,17].

Antimicrobial Assays of Pure Compounds
Bacterial strains (S. aureus ATCC 43300 (MRSA), E. coli ATCC 25922, P. aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700603, Acinetobacter baumannii ATCC 19606) were cultured in either Luria broth (LB) (In Vitro Technologies, USB75852, Victoria, Australia), nutrient broth (NB) (Becton Dickson, 234,000, New South Wales, Australia) or MHB at 37 • C overnight [5,18]. A sample of culture was then diluted 40-fold in fresh MHB and incubated at 37 • C for 1.5−2 h. The compounds were serially diluted 2-fold across the wells of 96-well plates (Corning 3641, nonbinding surface), with compound concentrations ranging from 0.015 to 64 µg/mL, plated in duplicate. The resultant mid log phase cultures were diluted to the final concentration of 1 × 10 6 CFU/mL; then, 50 µL was added to each well of the compound containing plates, giving a final compound concentration range of 0.008-32 µg/mL and a cell density of 5 × 10 5 CFU/mL. All plates were then covered and incubated at 37 • C for 18 h. Resazurin was added at 0.001% final concentration to each well and incubated for 2 h before MICs were read by eye.
Fungi strains (Candida albicans ATCC 90028 and Cryptococcus neoformans ATCC 208821) were cultured for 3 days on YPD agar at 30 • C. A yeast suspension of 1 × 10 6 to 5 × 10 6 CFU/mL was prepared from five colonies. These stock suspensions were diluted with yeast nitrogen base (YNB) (Becton Dickinson, 233,520, New South Wales, Australia) broth to a final concentration of 2.5 × 10 3 CFU/mL. The compounds were serially diluted 2-fold across the wells of 96-well plates (Corning 3641, nonbinding surface), with compound concentrations ranging from 0.015 to 64 µg/mL and final volumes of 50 µL, plated in duplicate. Then, 50 µL of a previously prepared fungi suspension, in YNB broth to the final concentration of 2.5 × 10 3 CFU/mL, was added to each well of the compound-containing plates, giving a final compound concentration range of 0.008-32 µg/mL. Plates were covered and incubated at 35 • C for 36 h without shaking. C. albicans MICs were determined by measuring the absorbance at OD 530 . For C. neoformans, resazurin was added at 0.006% final concentration to each well and incubated for a further 3 h before MICs were determined by measuring the absorbance at OD 570-600 .
Colistin and vancomycin were used as positive bacterial inhibitor standards for Gramnegative and Gram-positive bacteria, respectively. Fluconazole was used as a positive fungal inhibitor standard for C. albicans and C. neoformans. The antibiotics were provided in 4 concentrations, with 2 above and 2 below their MIC value, and plated into the first 8 wells of Column 23 of the 384-well NBS plates. The quality control (QC) of the assays was determined by the antimicrobial controls and the Z'-factor (using positive and negative controls). Each plate was deemed to fulfil the quality criteria (pass QC), if the Z'-factor was above 0.4, and the antimicrobial standards showed full range of activity, with full growth inhibition at their highest concentration, and no growth inhibition at their lowest concentration [5,18].

Cytotoxicity Assays
To a 384-well plate containing the 25× (2 µL) concentrated compounds, HEK-293 cells, counted manually in a Neubauer haemocytometer, were plated at a density of 5000 cells/well into each well [18,19]. The medium used was Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were incubated together with the compounds for 20 h at 37 • C, 5% CO 2 . Resazurin (5 µL (equals 100 µM final)) was then added to each well and incubated for further 3 h at 37 • C with 5% CO 2 . After final incubation, fluorescence intensity was measured as Fex 560/10 nm, em 590/10 nm (F 560/590 ) using a Tecan M1000 Pro monochromator plate reader. CC 50 values (concentration at 50% cytotoxicity) were calculated by normalizing the fluorescence readout, with 74 µg/mL tamoxifen as negative control (0%) and normal cell growth as positive control (100%). The concentration-dependent percentage cytotoxicity was fitted to a dose-response function (using Pipeline Pilot) and CC 50 values determined [18,19].

Haemolytic Assay
Human whole blood was washed three times with 3 volumes of 0.9% NaCl and then resuspended in the same solution to a concentration of 0.5 × 10 8 cells/mL, as determined by manual cell count in a Neubauer haemocytometer. The washed cells were then added to the 384-well compound-containing plates for a final volume of 50 µL. After a 10 min shake on a plate shaker, the plates were then incubated for 1 h at 37 • C. The plates were then centrifuged at 1000 g for 10 min to pellet cells and debris; 25 µL of the supernatant was then transferred to a polystyrene 384-well assay plate. Haemolysis was determined by measuring the supernatant absorbance at 405 mm (OD 405 ) using a Tecan M1000 Pro