Introduction

As a part of our ongoing natural product discovery initiative program, we are investigating soil actinomycetes collected near thermal vents emanating from a range of underground coal mine fire sites throughout Appalachia.1, 2 Notable alteration of soil composition in and around the vents due to the fire-related emission of greenhouse gases and the dispersion of volatile organic and inorganic species through surface vents of the mine, in conjunction with the natural biodiversity of Applachian Mountain, supports the contention that such collection sites present unique ecological environments for the soil actinobacteria-based natural product discovery.3, 4, 5, 6, 7, 8, 9, 10, 11 Located in the upper Elkhorn No. 3 coal bed of Appalachian Mountain of Eastern Kentucky, the Truman Shepherd coal bed was last mined in 1930s and the corresponding Truman Shepherd coal mine fire first observed in 2009.8 More than 70 pure bacterial cultures isolated from the soil samples collected around the vents of the Truman Shepherd fire displayed actinobacteria-like morphology on solid agar media. Comparison of HPLC—HR-MS molecular ions of the Streptomyces sp. TS-2-2 culture extracts to Antibase12 revealed the potential presence of novel metabolites, with the corresponding MS fragmentation patterns suggestive of glycosylated natural products. In this report, we describe the isolation, structure elucidation and biological activity of a new 20-membered glycosylated macrolide venturicidin C (1), along with the two known counterparts venturicidins A (2) and B (3), from a mycelial extract of Streptomyces sp. TS-2-2 (Figure 1 and Supplementary Figure S1). Also reported herein for the first time are the full NMR spectral data sets for venturicidins A (2) and B (3) to complement the previously published isolation/characterization data.13, 14, 15, 16, 17, 18, 19, 20 Nine additional known compounds namely nocardamine,21, 22 dehydroxynocardamine,21 desmethylenylnocardamine,21 ferrioxamine E,23, 24 adenosine, riboflavin, cyclo(D)-trans-4-OH-Pro-(D)-Phe,25 cyclo(D)-Pro-(D)-Phe25 and N-(2-phenylethyl)-acetamide26 were also isolated during the culture extract work-up (Supplementary Figure S46).

Figure 1
figure 1

Chemical structure of venturicidins C (1), A (2) and B (3), along with the related macrolides 4–8.

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Results and discussion

Streptomyces sp. TS-2-2 was fermented in liquid medium for both small-scale (50 ml) screening and large-scale (8 l) production of metabolites (Supplementary Figure S5). Screening of metabolites in low-resolution LC-ESI-MS revealed three compounds that displayed similar retention time, UV profile (Supplementary Figure S2) and MS fragmentation pattern. A difference of 231 AMU between the base MS ion and the fragmentation MS ion for compounds 1 and 2, and 170 AMU for compound 3 suggested a parental glycoside. The extract did not reveal any major UV–visible spots (at 254 or 365 nm) on TLC and treatment with anisaldehyde/sulfuric acid followed by heating revealed a spot with an intensive blue coloration that turned dark-green within a few minutes. This suggested a predominance of non-aromatic compounds within the extract. Subsequent purification of compounds from a large-scale fermentation extract using various chromatographic techniques led to the isolation of one new macrolide, venturicidin C (1, 3.2 mg l−1), along with the two known antifungal macrolide antibiotics venturicidin A (2; 40.0 mg l−1) and B (3; 10.8 mg l−1) (see EXPERIMENTAL SECTION and Supplementary information for details). The structure of these molecules was determined by a cumulative consideration of 1D and 2D NMR spectroscopy and HR-MS (ESI) data.

Structure elucidation

The physicochemical properties of compounds 1–3 are summarized in Table 1. Compound 1 was isolated as white powder (3.2 mg l−1) from the mycelial extract using various chromatographic techniques (Supplementary Figure S3). The molecular formula of 1 was deduced as C42H69NO11 on the basis of HR-ESI-MS (m/z 786.4799 [M+Na]+, calcd. 786.4763 for C42H69NO11Na) and 1H and 13C NMR. The HR-ESI-MS spectrum of 1 also revealed a fragmentation ion (m/z 555.4067) consistent with the cleavage of a glycosidic bond and the elimination of two water molecules (Figure 2 and Supplementary Figures S8 and S9). The proton NMR spectrum of 1 in CD3OD (Table 2) was rich in aliphatic proton signals with four additional olefinic signals. The two triplet signals observed at δ 1.00 and δ 0.98, five doublets at δ 1.27, 0.98, 0.91, 0.88 and 0.82 along with two singlets at δ 1.43 and 1.48, indicated the presence of nine methyl groups in the molecule. When the solvent was changed to dimethylsulfoxide (DMSO)-d6, an additional broad signal comprising two protons were observed at δ 6.48, suggesting the presence of an amino group (-NH2).

Table 1 Physicochemical properties of macrolides 1–3
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Figure 2
figure 2

ESI/MS fragmentation patterns of venturicidins C (1), A (2) and B (3).

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Table 2 1H NMR spectroscopic data of venturicidins C (1), A (2) and B (3)
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The 13C NMR/HSQC spectra of 1 (Table 3) displayed 42 signals, which corresponded to 9 methyl groups, 10 methylene groups, 6 quaternary carbons and 17 methine groups, of which 8 were oxygenated. In addition, carbon signals pertaining an acetal (δ 99.8), a ketal (δ 95.3), six olefinic (δ 140.1∼118.3), an amide (δ 159.7), a lactone/carboxylic acid/amide (δ 174.0) and a ketone (δ 218.0) functionality appeared in the downfield region of the spectrum. The presence of 3′-O-carbamoyl-β-D-olivose sugar moiety in compound 1 was confirmed through both the MS fragmentation pattern and the cumulative analyses of 1H-1H-COSY/HMBC spectra (Figure 3). The 3J HMBC correlation observed between H-3′ and the quaternary carbon at δ 159.7 confirmed the attachment of the C-3′ sugar carbamoyl group. A substructure search of AntiBase 201212 using the 3′-O-carbamoyl-β-D-olivose moiety as a prompt revealed only four 20-membered macrolides—venturicidin A (2),27, 28, 29 X-14952B (4),30 17-hydroxyventuricidin A (5),14 and irumamycin (6).31 Comparison of the 13C NMR chemical shifts of 1 with that reported for X-14952B (4) revealed both compounds to share an identical glycosylated macrolactone core with structural divergence at C-17 (Table 3). Specifically, compared with 4, 1 lacks a C-17 hydroxyl as indicated by a notable C-17 13C NMR chemical shift (δ 43.8 in 1 compared with δ 78.2 in 4). The 3J HMBC cross-peaks observed between H-13 and C-1′ (δ 99.8) and between H-1′ and C-13 (δ 84.2) are consistent with the attachment of 3′-O-carbamoyl-β-D-olivose sugar moiety at C-13 position of 1 as previously observed for 2 and 4–6.14, 30, 31 All of the remaining HMBC correlations (Figure 3) and NMR data (Tables 2 and 3) are in full agreement with structure 1. The relative configuration at the stereocenters was indirectly established through the analyses of NOESY correlations (Figure 4) and their comparison with those of the reported analogs 2–9.16, 32 Thus, thorough analyses of 1H, 13C, HSQC, 1H-1H-COSY, TOCSY, HMBC and NOESY spectra cumulatively (Supplementary Figures S7 and S10–S21) established the structure of compound 1 as depicted in Figure 3 and was subsequently named as venturicidin C.

Table 3 13C NMR spectroscopic data of venturicidins C (1), A (2) and B (3) in comparison with the literature data of X-14952B (4)30 and 17-hydroxyventuricidin A (5), δ measured in p.p.m.
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Figure 3
figure 3

Selected 1H,1H-COSY (—) and HMBC (→) correlations of venturicidin C (1).

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Figure 4
figure 4

Selected NOESY () couplings of venturicidin C (1).

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The physicochemical properties of compound 2 (40.0 mg l−1) were similar to those of 1. Low-resolution ESI MS revealed a 749 Da base peak suggesting its molecular weight as 14 AMU less than that of 1.The molecular formula of compound 2 was deduced as C41H67NO11 based on the quasi-molecular ion peak observed at m/z 772.4676 [M+Na]+ in HR-ESI-MS spectrum. The presence of an additional fragment peak at m/z 541.3908 was consistent with the generation of an aglycone fragment via the elimination of the sugar moiety and two water molecules (Figure 2 and Supplementary Figures S22 and S23). Like 1, the proton NMR spectrum of 2 indicated nine methyl groups. The methyl triplet at δ 0.98 in 1 (24-CH2CH3) was missing in case of 2, and instead, a methyl doublet was displayed at δ 0.96 in 2 (24-CH3). The 13C NMR spectrum revealed 41 signals. The chemical shift in the sugar residue and the lactone part of the aglycone moiety in 2 were nearly identical to those of 1 in both CD3OD and DMSO-d6 solvents (Tables 2 and 3). Thorough analyses of NMR spectra indicated 2 and 1 to share an identical 20-membered lactone core with the sole structural difference C-24 (-CH2CH3 in 1 versus -CH3 in 2). A substructure search in AntiBase 2012 revealed the identity of 2 as venturicidin A, also known as aabomycin A1 (2). Although this molecule was isolated previously from several Streptomyces species (including Streptomyces sp. no. 325-17, Streptomyces griseolus, Streptomyces xanthophaeus and Streptomyces aureofaciens) and its structure subsequently determined through chemical degradation and through partial NMR analyses, we report here for the first time the complete physicochemical and NMR spectroscopic data (Figures 5 and 6, Supplementary Figures S24–S33 and Tables 1, 2, 3).27, 28, 29

Figure 5
figure 5

Selected 1H,1H-COSY (—) and HMBC (→) correlations of venturicidin A (2).

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Figure 6
figure 6

Selected NOESY () couplings of venturicidin A (2).

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Likewise, compound 3 was isolated as white powder (10.8 mg l−1) and displayed similar physicochemical properties to that of 1 and 2. ESI-MS spectrum of 3 revealed a molecular weight of 706, 43 AMU less than that of 2. The molecular formula of 3 was deduced as C40H66O10 (m/z 729.4594 [M+Na]+ with an identical fragment peak at m/z 541.3915 as 2, indicating the same aglycon in both compounds (Figure 2 and Supplementary Figures S34 and S35). This implicated structural divergence of the appended sugar wherein the 43 AMU molecular weight difference between 3 and 2 could correlate to the sugar C-3′ amide group (-CONH2). Consistent with this, the carbamoyl carbon signal observed at δ 159.6 in the 13C NMR spectrum of compound 2 was absent in the spectrum for 3. Thus, through the cumulative analysis of NMR data and relevant precedent in the literature, the structure of compound 3 was confirmed as depicted in Figures 7 and 8—the known molecule venturicidin B (aabomycin A2). Although venturicidin B was previously reported as a metabolite of S. aureofaceins,13 we report here for the first time the complete physicochemical and NMR spectroscopic data (Supplementary Figures S34–S45).

Figure 7
figure 7

Selected 1H,1H-COSY (—) and HMBC (→) correlations of venturicidin B (3).

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Figure 8
figure 8

Selected NOESY () couplings of venturicidin B (3).

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Including ventruricidin C reported herein, the venturicidins make up 9 of the 17 naturally occurring 20-membered macrolide glycosides. The closely related congeners venturicidins A,13 B,13 X,16 X-14952B,30 17-hydroxyventuricidin A,14 irumamycin,31 3′-decarbamoylirumamycin14 and irumanolide II32 are summarized in Figure 1. Their remarkable potency against different fungal strains14, 15 and their antitrypanosomal activities18 are notable. Consistent with this precedent, the antifungal activities of venturicidins A, B and C against Cladosporium cucumerinum ATCC 26212 were comparable in disc diffusion assays (Supplementary Figure S48). Mechanistically, venturicidins inhibit F0F1-ATPase and are also known to inhibit ATP synthesis in both fungi and bacteria.17, 19, 33 However, unlike their structurally/mechanistically similar cytotoxic counterparts (ossamycin, cytovaricin and apoptolidin),34 venturicidins A, B or C did not exhibit significant cytoxoxicity against non-small-cell carcinoma cell line A549 (Supplementary Figure S47). This distinction is noteworthy in the context of considering the further development of non-toxic anti-infective members of this unique family. None of these molecules exhibited antibacterial activities against bacterial test strains S. aureus ATCC 6538 and S enterica ATCC 10708 up to 124 μM concentrations.

Experimental section

General experimental procedures

UV spectra were recorded on an Ultrospec 8000 spectrometer (GE, Pittsburgh, PA, USA). NMR spectra were measured on a Varian VnmrJ 500 (Varian, Palo Alto, CA, USA; 1H, 500 MHz; 13C, 125.7 MHz) and VnmrJ 400 (1H, 399.8 MHz; 13C, 100.5 MHz) spectrometers; the δ-values were referenced to the respective solvent signals. ESI mass spectra were recorded on a Finnigan LCQ ion trap mass spectrometer (Thermo Finnigan, Waltham, MA, USA). HR-ESI mass spectra were recorded on AB SCIEX Triple TOF 5600 System (AB Sciex, Framingham, MA, USA). HPLC-MS analyses were carried out in Waters 2695 LC module (Waters, Milford, MA, USA) using a Symmetry Anal C18 5 μm column (4.6 × 250 mm; Waters) and a gradient elution profile (solvent A: H2O; solvent B: acetonitrile; flow rate: 0.5 ml min−1; 0–4 min, 90% A and 10% B, 4–22 min, 90–0% A, 22–27 min, 0% A and 100% B, 27–29 min, 0–90% A, 29–35 min, 90% A and 10% B). For preparative scale separation, Phenomenex (Torrance, CA, USA) C18 column (10 × 250 mm, 5 μm) was used on a Varian ProStar Model 210 equipped with a photodiode diode array detector and a gradient elution profile (solvent A: H2O; solvent B: acetonitrile; flow rate: 5.0 ml min−1; 0–2 min, 75% A and 25% B, 2–15 min, 75–0% A, 15–17 min, 0% A and 100% B, 17–18 min, 0–75% A, 18–19 min, 75% A and 25% B). All solvents used were of ACS grade and purchased from the Pharmco-AAPER (Brookfield, CT, USA). Silica gel (230–400 mesh) for column chromatography was purchased from Silicycle (Quebec City, QC, Canada). Rf values were measured on Polygram SIL G/UV254 (Macherey-Nagel, Dueren, Germany). Amberlite XAD-16 was obtained from Sigma-Aldrich (St Louis, MO, USA). Size exclusion chromatography was performed on Sephadex LH-20 (25∼100 μm; GE Healthcare, Piscataway Township, NJ, USA).

Isolation of Streptomyces sp. TS-2-2 and its taxonomy

A soil sample containing TS-2-2 was collected from the Truman Shepherd underground mine fire, Floyd County, KY, USA (coordinates: N 37° 28.218′ and W 83° 51.132′). Streptomyces sp. TS-2-2 was isolated following previously reported methods.35 Genomic DNA was isolated from a fully grown colony using InstaGene Matrix (Bio-Rad, Hercules, CA, USA). DNA was purified using QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). The partial 16S rRNA gene fragment was amplified using universal primers (27F, 5′-AGAGTTTGATCMTGGCTCAG-3′; 1492R, 5′-GGTTACCTTGTTACGACTT-3′) and Advantage GC2 DNA polymerase (Clontech, Mountain View, CA, USA).36 Polymerase chain reaction conditions were as follows: initial denaturation for 95 °C for 3 min followed by 30 cycles at 94 °C for 30 s, 48 °C for 30 s, 68 °C for 1 min 30 s, followed by a final extension temperature at 68 °C for 5 min. QIAquick gel extraction kit (Qiagen) was used to gel purify the amplified product. The amplified fragment was sequenced (1256 bp) and the sequence was used to search the NCBI 16S rRNA library for bacteria and archaea by Basic Local Alignment Search Tool (BLAST). This comparison revealed a 99% identity to the 16S rRNA gene sequence of S. bingchenggensis BCW-1. The sequence of 16S rRNA has been deposited in the NCBI nucleotide database under the accession number KC526988.

Cell viability assay

Conversion of resazurin (7-hydroxy-10-oxido-phenoxazin-10-ium-3-one) to its fluorescent product resorufin was monitored to assess viability of human lung non-small-cell carcinoma cell line A549. DMEM/F-12 Kaighn’s modification and MEM/EBSS media (Thermo Scientific, Rockford, IL, USA) were used to grow A549 (ATCC, Manassas, VA, USA) with 10% heat-inactivated fetal bovine serum, 100 U ml−1 penicillin, 100 μg ml−1, streptomycin and 2 mM L-glutamine. Cells were seeded at a density of 2 × 103 cells per well in 96-well clear bottom culture plates (Corning, NY, USA), incubated 24 h at 37 °C in a humidified atmosphere containing 5% CO2 and were subsequently exposed to known toxin (1.5 mM hydrogen peroxide, 10 μg ml−1 actinomycin D) and test compounds for 2 days. To assess cell viability, 150 μM of resazurin (Sigma, St Louis, MO, USA) was added to each well, plates were shaken briefly for 10 s and incubated for another 3 h (A549 cells) at 37 °C to allow viable cells to convert resazurin into resorufin. The fluorescence intensity for resorufin was detected on a scanning microplate spectrofluorometer FLUOstar Omega (BMG Labtech, Cary, NC, USA) using an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The assay was repeated in three independent experiment replications. In each replication, the resorufin values of treated cells were normalized to, and expressed as percent of, the mean resorufin values of untreated, metabolically active cells (100%, all cells are viable). In addition, the CellTox Green Cytotoxicity Assay (Promega, Madison, WI, USA) was applied for assessing the level of dead cells, or cytotoxicity, in the same A549 cell culture. Before assaying viability with resazurin, 20 μl of CellTox Green Dye (a proprietary asymmetric cyanine dye) reagent (Promega Corporation) was added to each well of plate with cells, plate was mixed on shaker for 1 min, incubated for 15 min at room temperature and fluorescent intensity of dye intercalated with dead cells DNA was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm on a scanning microplate spectrofluorometer FLUOstar Omega (BMG Labtech), and then resazurin was added and viability was assayed as described above. Cytotoxicity values were converted to corresponding viability values and plotted on the same graph with resazurin assay data.

Media, fermentation and isolation

A-medium

Glucose (10.0 g), yeast extract (5.0 g), soluble starch (20.0 g), peptone (5.0 g), NaCl (4.0 g), K2HPO4 (0.5 g), MgSO4·7H2O (0.5) and calcium carbonate (2 g) were dissolved in 1 liter of demineralized water. The suspension (pH 7.0) was sterilized by autoclaving for 33 min at 121 °C.

M 2 agar plates

Glucose (4.0 g), malt extract (10.0 g), yeast extract (4.0 g) and agar (15.0 g) were dissolved in 1 liter of demineralized water. The suspension (pH 7.2) was sterilized by autoclaving for 33 min at 121 °C.

Fermentation, extraction and isolation

The terrestrial Streptomyces sp. TS-2-2 was cultivated on M2 agar plates at 28 °C for 3 days. To prepare the seed culture, chuncks of grown agar plate were used to inoculate three 250-ml baffled flasks, each containing 100 ml of A-medium, and the cultures grown at 28 °C with shaking (210 r.p.m.) for 3 days. An aliquot of seed culture (3 ml) was subsequently used to inoculate 80–250 ml baffled flasks, each containing 100 ml of A-medium. Fermentation was continued at 28 °C with shaking (210 r.p.m.) for 5 days. The obtained reddish-brown culture broth was centrifuged and filtered over celite. The supernatant was mixed with XAD-16 (4%) resin overnight, followed by filtration. The resin was washed with water (4 × 800 ml) and then extracted with methanol (3 × 600 ml). The methanol extract was subsequently evaporated in vacuo at 38 °C to afford 15.1 g of reddish-brown solid crude extract. The biomass (mycelium) was extracted with methanol (3 × 500 ml) followed by acetone (1 × 800 ml) and then evaporated in vacuo at 38 °C to yield 8.8 g of reddish-brown solid crude extract. Both extracts revealed different sets of metabolites based on HPLC and TLC analyses wherein the targeted metabolites were found to be mostly present in the mycelium extract. Thus, this extract was subjected to the following work-up and isolation procedure.

The mycelial extract (8.8 g) was dissolved in MeOH/50% H2O (40 ml) and was fractionated with a gradient of H2O/0–100% CH3CN using RP-18 column (column 3 × 30 cm, 75 g) chromatography. This resulted in the generation of following fractions: 0.25 l 0% CH3CN and 0.25 l 10% CH3CN→fraction FI (1.6 g); 0.25 l 20% CH3CN→fraction FII (0.8 g); 0.25 l 30% CH3CN→fraction FIII (0.6 g); 0.25 l 40% CH3CN→fraction FIV (0.25 g); 0.25 l 60% CH3CN, 0.25 l 50% CH3CN, 0.25 l 25% CH3CN (combined based on the HPLC and TLC similarity)→fractions FV-FVII (1.2 g); 0.5 l 100% CH3CN→fraction FVIII (0.92 g). TLC analysis followed by anisaldehyde sulfuric acid development indicated that the target dark-green band was present mainly in fraction FVIII (0.92 g) (see Supplementary Figure S4). Further purification of fraction FVIII using sephadex LH-20 (CH2Cl2/40% MeOH, 2.5 × 50 cm) and then by HPLC (see Supplementary Figure S6 for HPLC conditions) afforded venturicidins A (2; 40.0 mg ml−1), B (3; 10.8 mg ml−1) and C (1; 3.2 mg ml−1) as white powders. Fractions FI, FII and FV-FVII were avoided based on the HPLC/MS and TLC analysis, as no related compounds were observed. Further purifications of fractions FIII and FIV using Sephadex LH-20 (MeOH, 2.5 × 40 cm) led to the isolation of nocardamine–Fe complex (240.0 mg, red solid, molecular weight 654 Da) and ferrioxamine E (45.8 mg, orange solid), respectively.

Similarly, the XAD-16 extract (15.1 g) was dissolved in MeOH/50% H2O (60 ml) and then subjected to fractionation using RP-18 column (column 3 × 30 cm, 75 g) chromatography and gradients of H2O/0–100% CH3CN systems. This resulted in the generation of nine fractions as follows: 0.25 l 0% CH3CN and 0.25 l 10% CH3CN→fraction FI (5.5 g); 0.15 l 20% CH3CN→fraction FII (1.3 g); 0.25 l 20% CH3CN→fraction FIII (1.25 g); 0.25 l 30% CH3CN→fraction FIV (0.65 g); 0.25 l 40% CH3CN→fraction FV (2.5 g); 0.25 l 50% CH3CN→fraction FVI (0.20 g); 0.25 l 60% CH3CN→fraction FVII (0.15 g); 0.25 l 80% CH3CN→fraction FVIII (0.55 g); 0.5 l 100% CH3CN→fraction FIX (0.21 g). Fractions FI and FIX were discarded as no significant metabolites were observed by TLC or HPLC/MS. Fractions FII, FIII and FIV were combined based on their HPLC/MS and TLC similarities. Further purification of the combined fractions using Sephadex LH-20 and HPLC afforded adenosine (131.2 mg, white powder), riboflavin (120.8 mg, yellowish-green solid) and cyclo(D)-trans-4-OH-Pro-(D)-Phe (28.3 mg, colorless solid).25 In a similar manner, fractions FVI and FVII were combined and purified to afford cyclo(D)-Pro-(D)-Phe25 (29.8 mg, colorless solid) and N-(2-phenylethyl)-acetamide26 (10.8 mg, colorless solid) in pure forms. HPLC purification of fraction FIII (0.55 g) led to the isolation of nocardamine21, 22 (28.3 mg), dehydroxynocardamine21 (4.8 mg) and desmethylenylnocardamine21 (3.7 mg) as white solids. Fine crystals were observed in fraction FV (2.5 g) when it was kept overnight in H2O/40% CH3CN. Filtration of fraction of crystals followed by washing with water afforded nocardamine (1.6 g, white needles). The filtrate was not further analyzed as it mainly contained an unknown nocardamine–Fe complex23 (1.31 g, red solid, molecular weight 654 Da) (Supplementary Figure S3).

Antibacterial and antifungal activity tests

The fungal strain S. cerevisiae (ATCC 204508) and the two bacterial strains S. aureus (ATCC 6538) and S. enterica (ATCC 10708) were used as model strains for susceptibility assays. S. cerevisiae, S. enterica and S. aureus were grown in liquid or on agar plates using YAPD (ATCC medium number 1069), nutrient broth (BD 234000) and in tryptic soy broth (BD 211825) media, respectively. Individual strains were grown in 5 ml medium for 16 h at 37 °C with shaking (200 r.p.m.). An aliquot of a fully grown culture (100 μl) was diluted to 30 ml using sterile liquid medium. Aliquots (160 μl) of each diluted culture were then transferred into 96-well plates supplied with 2 μl of venturicidins (final concentration of range 1–125 μm). Negative controls tested in parallel include vehicle alone (DMSO) while positive controls ampicillin and kanamycin sulfate (200 μg each for S. enterica and S. aureus) or cycloheximide (50 μg for S. cerevisiae) were used. The culture plate was incubated at 37 °C for 16 h with shaking (200 r.p.m.) and the OD600 (optical density at wavelength 600 nm) was subsequently measured using a scanning microplate spectrofluorometer FLUOstar Omega (BMG Labtech). The acquired OD600 values were normalized considering the negative control wells have 100% viability.

The fungal strain C. cucumerinum was used in disc diffusion assays. Solutions of amphotericin B and test compounds were made in DMSO. Each sterile paper disc was loaded with 20 μl solution and was allowed to dry in the biosafety cabinet for 4 h. The dried discs were then placed on the V8 agar plate following the homogeneous distribution of C. cucumerinum spores. DMSO was used as a negative control. The plates were then incubated at 24 °C for 3 days under the dark condition. Inhibition zone were then measured.

V8-medium

Six grams of calcium carbonate was added into 360 ml V8 juice and stirred the mixture for 30 min. Supernatant liquid was collected following centrifugation at 4000 g for 10 min. pH of the solution was adjusted to 7.0 using 1 M KOH solution. The solution was then diluted to fourfold with water. Bacto agar was added to 1.5% (w v−1) and then autoclaved to prepare V8 agar plates.