Fermentation and extraction

A 2 mL frozen glycerol stock of strain CNT-373 was used to inoculate 25 mL of liquid A1 medium and shaken at 230 rpm and 27°C. After 5 days, the seed culture was used to inoculate a 1 L culture in growth medium A1bfe+C (A1 medium with the addition of 1 g CaCO₃, 100 mg KBr and 40 mg Fe₂(SO₄)₃•4H₂O). After 3 days of shaking, 25 mL aliquots were used to inoculate 18 Fernbach flasks (2.8 L) each containing 1 L medium A1bfe+C. After 7 days of cultivation, sterilized XAD-16 resin (20 g per L) was added, shaken for 6 hours, and collected by filtration through cheesecloth. The resin was then washed with deionized water and eluted with acetone. The acetone solvent was removed under reduced pressure and the resulting aqueous layer extracted with ethyl acetate (3 × 500 mL). The combined ethyl acetate extracts were dried over anhydrous sodium sulfate, decanted, and concentrated to dryness to yield 2.4 g of crude material.

Isolation of nosiheptide

The crude extract was dissolved in methanol and dichloromethane, adsorbed onto silica gel (2.5 g), and fractionated on a short column of silica gel (6 × 2.5 cm, h × w) using a 10% step gradient from 100% isooctane to 100% ethyl acetate as eluents). Fractions with antibacterial activity were eluted with 90% and 100% ethyl acetate. The active fractions were pooled and subjected to a size exclusion column chromatography with Sephadex LH-20 using methanol as eluent. The final purification step was achieved with silicia gel, using ethyl acetate with 10% dimethylformamide (DMF) to give nosiheptide (210 mg).

Spectroscopic analysis of nosiheptide

¹H, ¹³C and 2D NMR spectroscopic data were obtained on a Varian Inova 500 MHz spectrometer in a solvent mixture of methanol-d₄ and CDCl₃ (3:1) to facilitate solubility. Offline processing was conducted using topspin NMR software by Bruker BioSpin 2011 (iNMR, http://www.inmr.net). The NMR data (Table 1) is in good agreement with previously published NMR data for nosiheptide,⁸ with the resultant nosiheptide structure shown (Figure 1). High resolution ESI-TOF mass spectra were provided by the mass spectrometry facility at the Department of Chemistry and Biochemistry at the University of California San Diego, CA. HR-ESI-TOF-MS [M+H]⁺ m/z 1222.1565 (calcd for C₅₁H₄₄N₁₃O₁₂S₆ 1222.1551, Δ1.13 ppm). Low resolution LC-MS data were measured using a Hewlett-Packard HP1100 integrated LC/MS system with a reversed-phase C₁₈ column (Phenomenex Luna, 4.6 mm × 100 mm, 5 mm) at a flow rate of 0.7 mL/min.

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Figure 1

Structure of marine-derived nosiheptide.

NMR data in good agreement with:

Mocek, U., Chen, L.-C., Keller, P. J., Houck, D. R., Beale, J. M. & Floss, H. G. 1H and 13C NMR assignment of the thiopeptide antibiotic nosiheptide. J. Antibiot. 11, 1643-16-48 (1989).

Bacterial Strains and Susceptibility Testing

Details on the strains used in this study are in Table 2. MRSA Sanger 252 USA 200 strain was obtained through the Network of Antimicrobial Resistance in S. aureus (NARSA) program supported under NIAID/NIH contract # HHSN272200700055C. Susceptibility testing was performed in duplicate using cation-adjusted Mueller-Hinton broth (CA-MHB) and Mueller-Hinton agar (MHA) according to CLSI methods.⁹ The MIC for Clostridium difficile was determined by the agar dilution reference method according to CLSI guidelines.^10,11 Susceptibility of nosiheptide against MRSA strain TCH1516 was also determined in the presence of 20% activated-pooled human serum (freshly collected from consenting healthy donors) and 80% MHB by adding resazurin and assessing the conversion to resorufin exactly as previously described.¹² This method provides a visual readout of color change from the blue indicator resazurin to the pink resorufin, a sign of bacterial growth.

Time Kill Studies

Time kill studies were done essentially as previously described in this laboratory.¹³ Briefly, nosiheptide was added at multiples of the MIC in CA-MHB. MRSA strains or vancomycin-resistant Enterococcus faecium (VRE-CUS) were added at a starting inoculum of 5 × 10⁵ cfu/mL, and the cultures were incubated in a 37°C shaking incubator (New Brunswick Scientific, Enfield, CT). Samples were taken at specified timepoints for enumeration of viable bacteria by plating serial dilutions on Todd-Hewitt agar plates.

Post-Antibiotic Effect

Post-antibiotic effect was assessed using the viable plate count method as previously described.¹³ For these studies, either nosiheptide or vancomycin was added at 10X their respective MICs to 14 mL Falcon tubes containing CA-MHB. CA-MRSA strain TCH1516 or HA-MRSA strain Sanger 252 was added at 5 × 10⁵ cfu/mL, and the tubes were incubated under shaking conditions at 37°C for 1 hour. The bacteria were then pelleted and washed in 4 mL of antibiotic-free CA-MHB. The pellets were resuspended in 4 mL CA-MHB and incubated at 37°C in a shaking incubator. Surviving bacteria were assessed at specified timepoints as for the time-kill assay. The PAE was determined as previously described.^13,14

Mammalian Cell Cytotoxicity

Mammalian cell cytotoxicity was assessed as previously described.¹³ Briefly, 2 × 10⁴ HeLa cells (American Type Culture Collection # CCL-2, ATCC, Manassas, VA) were seeded per well of sterile 96-well tissue culture-treated plates (Falcon; Becton Dickinson, Franklin Lakes, NJ). After 24 hours, the medium (RPMI containing 10% heat-inactivated fetal bovine serum) was replaced with fresh medium containing increasing concentrations of nosiheptide up to 128 mg/L, and the plates were incubated at 37°C in 5% CO₂. Cell viability was assayed at 72 hours by measuring the reduction of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] using the CellTiter 96 Aqueous nonradioactive cell proliferation assay according to the manufacturer's instructions (Promega, Madison, WI).

In Vivo Testing

A murine model of intraperitoneal (ip) infection was used to test in vivo efficacy of nosiheptide.¹⁵ Eight week old female CD1 mice (Charles River, Wilmington, MA, USA) were injected ip with 1 – 2 × 10⁹ cfu of the HA-MRSA strain Sanger 252 in 4% hog gastric mucin. The mice were treated with nosiheptide (20 mg/kg) ip at 1 and 8 h after bacterial inoculation and were monitored for mortality and clinical status twice daily thereafter for five days. Moribund mice were humanely euthanized as were mice at the end of the study. These experiments were reviewed and approved by the Animal Subjects Committee of UCSD.

Time-kill kinetics

In vitro time-kill analysis was used to assess nosiheptide killing kinetics. Nosiheptide was rapidly bactericidal against MRSA in a concentration- and time-dependent manner (Figure 2A), with a nearly 2-log kill noted within 6 h at both 10X and 20X MIC. A 2-log kill of MRSA was also noted for nosiheptide against MRSA resistant to other antibiotics such as linezolid or erythromycin (Figure 2B). While nosiheptide demonstrated bactericidal activity against MRSA at 10X and 20X the MIC, it was bacteriostatic against a clinical bloodstream isolate of vancomycin-resistant Enterococcus faecium up to 64X MIC (Figure 2C). The MRSA killing kinetics of nosiheptide also compared favorably with vancomycin kinetics at 10X and 20X their respective MICs (Figure 2D).

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Figure 2

Time-kill kinetics of nosiheptide against MRSA and VRE. Nosiheptide killing kinetics was studied against MRSA and VRE at multiples of their MICs (MIC = 0.06 mg/L for nosiheptide). A.) Killing kinetics of nosiheptide at 1X, 10X, and 20X MIC against MRSA USA300 strain TCH1516 (MIC = 0.06 mg/L). B.) Nosiheptide killing kinetics against various multi-drug resistant MRSA at 10X their respective MICs. C.) Nosiheptide activity over time against VRE-CUS at multiples of the MIC (MIC = 0.125 mg/L). D.) Nosiheptide time-kill kinetics against MRSA USA300 strain TCH1516 compared to vancomycin at 10X and 20X their respective MICs (MIC = 0.06 mg/L for nosiheptide; MIC = 1.56 mg/L for vancomycin). Each assay was repeated in duplicate (for VRE) or triplicate (for MRSA), and the data shown are the mean +/− SD from one representative experiment.

Prolonged post-antibiotic effect and lack of cytotoxicity

Given its favorable MRSA killing kinetics, nosiheptide was assessed for its post-antibiotic effects (PAE). Notably, nosiheptide exhibited prolonged PAEs against both HA- and CA-MRSA compared to the most commonly used systemic antibiotic, vancomycin, at 10X their respective MICs (Figure 3A and B). The PAE for nosiheptide was calculated to exceed 9 h for both HA- and CA-MRSA. We also tested nosiheptide for in vitro evidence of mammalian cell cytotoxicity as a prelude to our in vivo experiments. No evidence for nosiheptide cytotoxicity was observed when incubated for 72 hours with the cervical carcinoma HeLa cell line at up to 128 mg/L, which is approximately 1000-fold above the MIC against MRSA. We also found that nosiheptide activity against USA300 MRSA was not inhibited in 20% human serum (Table 2).

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Figure 3

Post-antibiotic effect of nosiheptide on contemporary MRSA strains. CA-MRSA strain TCH-1516 (Panel A) or HA-MRSA strain Sanger 252 (Panel B) were incubated for one hour with nosiheptide or vancomycin at 10X MIC. The antibiotics were removed, and the bacteria were washed and allowed to recover in antibiotic-free media. The curves show the rates of growth following antibiotic treatment. The data represent the mean +/− SD of one representative assay repeated in duplicate.

N.M.H. was supported by the National Institutes of Health (NIH) Training Program in Marine Biotechnology (T32 GM067550) and a Ruth L. Kirschstein National Research Service Award (NRSA) from National Institutes of Health grants (5 F31 GM090658-02). S.L. was supported by the German Research Foundation (DFG). W.F., V.N., P.R.J., and M.E.H. were supported by National Institutes of Health grant GM084350. PRJ and WF acknowledge financial support from the NIH Fogerty Center International Cooperative Biodiversity Groups program (grant U01-TW00007-401). We thank K. Freel and C. Kauffman for assistance with fieldwork and W. Aalbersberg (University of the South Pacific) for providing laboratory space and facilitating the field collections. We gratefully acknowledge the people of Fiji for their hospitality and permission to collect samples from their local waters. We are also grateful to Diane M. Citron and Ellie Goldstein MD of R.M. Alden Research Laboratories (Culver City, CA) for performing susceptibility testing against Clostridium difficile.