Abstract The search for novel antibacterial agents has acquired a new sense of urgency due to the dramatic rise of antibiotic resistance among bacterial pathogens. This resistance is partly due to the poor structural diversity of the antibiotics used in chemotherapy: only a dozen of chemical scaffolds attacks a dozen of different target proteins. To increase structural diversity, we are currently exploring a bacterial collection to reach new antibacterial molecules. Thus, we have found a new antibacterial molecule, named margaucin, from a Bacillus sp. The structure of margaucin was elucidated by a combination of NMR and MS spectrometry analysis. It corresponds to a terpen derivative, and it has a MW of 210 g/mol. This molecule was active against Gram positive bacteria such as Staphylococcus, Enterococcus, Streptococcus, Bacillus (MIC = 3 µg/ml) including multiresistant staphylococcus clinical strains. No cytotoxic activity against MCF7 (breast tumoral cell) at doses up to 100 µg /ml and no in vivo toxicity against mouse at dose up to 100 mg/kg were observed. On Staphylococcus aureus septicaemia model, mouse (OF1 female) were protected at 100 mg/kg. Introduction In our screening program, we found antibiotic activity against Gram positive bacteria in the culture supernatant of the Bacillus sp strain JPL84. This poster describes the fermentation of this strain, the isolation, and the chemical as well as the biological characterization of the active compound: a terpen derivative called margaucin. Methods Producing organism Bacillus sp JPL84 was isolated by our team in 2005 from a soil sample from Agoût, France. The genus was determined by partial 16S rDNA analysis. The most related sequences were searched using the blast of the National Center for Biotechnology Information (NCBI). The strain was maintained at 4°C on an agar slant Casitone Yeast Extract agar medium (CYE agar) containing casitone 10g, yeast extract 1g, CaCl 2 1 g, and agar 14 g in 1 liter of tap water. Fermentation Fermentation was carried out in 10 liters of CYE medium containing peptone 100 g, yeast extract 10g, and CaCl 2 l0g at 28°C with aeration and agitation. An overnight culture (100 ml) in the same medium was used for seeding. The antibiotic production started at 14 h after the inoculation, then gradually increased and reached a maximum at h. The antibiotic production was controlled and quantified by diffusion test agar against Staphylococcus aureus CIP and by analytical HPLC. Purification Acetonitrile (10% v/v) and the adsorber resin Amberlite XAD-16 were added to the culture broth, and agitated for 12 h at 4 °C. The XAD beads were separated from the culture broth, washed with water and water/methanol (50/50) respectively, and eluted with methanol (100%). The eluate was concentrated by evaporation under reduced pressure. The margaucin was finally purified by reverse phase HPLC using a preparative C18 column (symmetryshield C18) and a linear gradient of H 2 O, 0.1% TFA -acetonitrile, 0.1% TFA from 20% to 80% in 30 mn at a flow rate of 10 ml/mn. After freeze drying lyophilisation, 30 mg of margaucin were obtained. Margaucin, a New Terpen Derivative Active Against Multiresistant Gram Positive Bacteria F2-502 Maxime Gualtieri 1, Laurence Charles 2, Gaëtan Herbette 3, Lionel Bastide 4, Philippe Villain-Guillot 1, and Jean-Paul Leonetti 1 1 CNRS UMR 5160, Centre de Pharmacologie et Biotechnologie pour la Santé, Faculté de Pharmacie, Montpellier, France; 2 JE 2421 TRACES, Aix-Marseille Université, Campus scientifique de Saint Jérôme, Marseille; 3 Spectropole, Campus scientifique de Saint Jérôme, Marseille, France; 4 Selectbiotics, Nîmes, France Contact information Maxime Gualtieri (+33) Results Structure elucidation The molecular formula was established as C 12 H 18 O 3 by a combination of spectroscopic techniques: one and two-dimensional NMR experiments and electrospray mass spectrometry. The 13 C, DEPT, 1 H, COSY, HMQC, and HMBC data were recorded in CD 3 OD with a Bruker Avance DPX-300 equiped with a 5 mm QNP direct probe operating at 300 MHz for 1H and 75 MHz for 13 C and are presented in Table 1. ESI-MS experiments revealed that the molecular weight of margaucin is 210, since a pseudo-molecular ion, [M+H]+, was detected at 211. The 13 C NMR spectra displayed 5 signals (40.4, 71.6, 95.6, 105.0, and ppm) and 1H NMR spectra displayed two groups of signals: First group with 3 1 H signals (2.66, 3.63, 5.81 ppm) correspond to proton bounded to carbon with visible resonance on 13 C spectra. All signals are singlets. Second group with 2 1 H signals (3.62, 5.79 ppm), with chemical shifts almost identical to the first group, bounded to carbon with non visible resonance on 13 C spectra. They correspond to an alcohol form of margaucin. An unshielded methylen group at 3.63 ppm was attributed to the presence of the  position of an oxygen atom and a carbonyl group. The COSY spectrum did not show any correlations, while results showed that protons are in a position of a quaternary carbon or an heteroatom. The presence of two quaternary carbons (170.2 and ppm), one methine (95.6 ppm), one methylene (71.6 ppm), and two equivalents of methyl groups (40.4 ppm) were deduced from the DEPT spectrum. The assignment of the connected protons to these carbons by 13 C- 1 H bond coupling was realized by the HMQC spectrum. The margaucin structure was confirmed by the long-range scalar interaction HMBC spectrum with cross peaks between C1/H3, C2/H3, and C2/H5 as shown in Fig 1. Combination of NMR data with molecular weight information from the electrospray mass spectrum strongly suggested a symmetric molecule. MS/MS experiments performed on the protonated molecule, [M+H]+, at m/z 211 gave rise to a fragmentation pattern which is consistent with the structure proposed in Figure 1 (daughter ions at m/z 193, 175, 169, 165, 153, 151, 147, 141, 133, 129, 123, 119, 111, 109, 105, 95, 93, 91, 85, 83, 81, 79, 71, 69, 67, 65, 57, 55, 43). The UV absorption spectrum revealed two maxima - max: 269 and 330. Biological properties The antimicrobial activity of margaucin is shown in Table 1. MICs were determined as recommended by the CLSI.This antibiotic was only active against Gram positive bacteria (Table 1) including multi-resistant strains (Table 2); nevertheless, it was also strongly active against Escherichia coli TolC, a Gram negative strain deficient in a multidrug efflux transporter. This result suggests that resistance of Gram negative bacteria to margaucin owes to the penetration barrier. No activity against Candida albicans, and no cytotoxic activity at doses up to 100 µg/ml in MCF7 (breast tumor cell) were observed. In vivo the molecule was not toxic in mice at doses up to 100 mg/kg. Using a Staphylococcus aureus smith septicaemia model, we observed a protection of the mouse (OF1 female) at 100 mg/kg. Conclusion This is the first report of a production of margaucin by a microorganism and of its antibacterial activity. However the large scale synthesis of a molecule identical to margaucin has already been described and is possible. This increases the potential interest of this molecule. Fig. 1. Structure of margaucin