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Margaucin, a New Terpen Derivative Active Against Multiresistant Gram Positive Bacteria
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Contact information
Maxime Gualtieri
(+33) 467 548 607
[email protected]
Maxime Gualtieri1, Laurence Charles2, Gaëtan Herbette3, Lionel Bastide4, Philippe Villain-Guillot1, and Jean-Paul Leonetti1
1CNRS
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; 4Selectbiotics, Nîmes, France
Table 1. Minimal Inhibitory Concentration
Abstract
Results
The search for novel antibacterial agents has acquired a new sense of urgency due to the dramatic rise of
Structure elucidation The molecular formula was established as C12H18O3 by a combination of spectroscopic
Organism
antibiotic resistance among bacterial pathogens. This resistance is partly due to the poor structural diversity of
techniques: one and two-dimensional NMR experiments and electrospray mass spectrometry. The 13C, DEPT,
the antibiotics used in chemotherapy: only a dozen of chemical scaffolds attacks a dozen of different target
1H,
proteins. To increase structural diversity, we are currently exploring a bacterial collection to reach new
5 mm QNP direct probe operating at 300 MHz for 1H and 75 MHz for 13C and are presented in Table 1. ESI-MS
Bacteria
COSY, HMQC, and HMBC data were recorded in CD3OD with a Bruker Avance DPX-300 equiped with a
antibacterial molecules. Thus, we have found a new antibacterial molecule, named margaucin, from a Bacillus
experiments revealed that the molecular weight of margaucin is 210, since a pseudo-molecular ion, [M+H]+,
sp. The structure of margaucin was elucidated by a combination of NMR and MS spectrometry analysis. It
was detected at 211.
corresponds to a terpen derivative, and it has a MW of 210 g/mol. This molecule was active against Gram
The 13C NMR spectra displayed 5 signals (40.4, 71.6, 95.6, 105.0, and 170.2 ppm) and 1H NMR spectra
positive bacteria such as Staphylococcus, Enterococcus, Streptococcus, Bacillus (MIC = 3 µg/ml) including
displayed two groups of signals:
multiresistant staphylococcus clinical strains. No cytotoxic activity against MCF7 (breast tumoral cell) at doses
First group with 3 1H signals (2.66, 3.63, 5.81 ppm) correspond to proton bounded to carbon with visible
up to 100 µg /ml and no in vivo toxicity against mouse at dose up to 100 mg/kg were observed. On
resonance on 13C spectra. All signals are singlets.
Staphylococcus aureus septicaemia model, mouse (OF1 female) were protected at 100 mg/kg.
Second group with 2 1H signals (3.62, 5.79 ppm), with chemical shifts almost identical to the first group,
MIC (µg/ml)
Staphylococcus aureus CIP 76.25
3.125
Staphylococcus epidermidis CIP 68.21
3.125
Enterococcus faecalis
3.125
Bacillus anthracis
Bacillus subtilis ATCC 27370
3.125
Escherichia coli CIP 76.24
> 100
Escherichia coli TolC
3.125
Salmonella typhi
> 100
Serratia marcescens
Pseudomonas aeruginosa CIP 76.110
> 100
6.25
> 100
Yeast
Candida albicans
> 100
bounded to carbon with non visible resonance on 13C spectra. They correspond to an alcohol form of
Table 2. Antibiotic susceptibility of the different Staphylococcus aureus clinical strains.
margaucin.
Introduction
In our screening program, we found antibiotic activity against Gram positive bacteria in the culture
An unshielded methylen group at 3.63 ppm was attributed to the presence of the a position of an oxygen atom
and a carbonyl group. The COSY spectrum did not show any correlations, while results showed that protons
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.
are in a position of a quaternary carbon or an heteroatom. The presence of two quaternary carbons (170.2 and
105.0 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 13C-1H 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
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
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 - lmax: 269 and 330.
on an agar slant Casitone Yeast Extract agar medium (CYE agar) containing casitone 10g, yeast extract 1g,
CaCl2 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 CaCl2 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 18-20 h. The antibiotic production was controlled and quantified by
Fig. 1. Structure
H3C
5
of margaucin
3
2
CH3
5
4
1
O
CH3
O
O
CH3
Staphylococcus aureus
H4
H7
H9
H1
H3
H16
H18
Penicillin G
R
R
R
R
R
R
R
Amoxicillin
R
R
R
R
R
R
R
Piperacillin
R
R
R
R
R
R
R
Oxacillin
S
S
R
S
S
R
R
Cefazolin
S
S
R
S
S
R
R
Tobramycin
S
S
R
S
S
R
R
Amikacin
S
S
R
S
S
R
R
Gentamicin
S
S
S
S
S
R
S
Tetracycline
S
S
S
-
-
R
-
Doxycycline
S
S
S
S
S
L
S
Erythromycin
S
R
S
R
R
R
R
Clindamycin
S
R
S
R
S
R
R
Pristinamycin
S
S
S
S
S
L
S
co-trimoxazole
S
S
S
S
S
S
S
Pefloxacin
-
-
R
R
S
-
R
Ofloxacin
S
R
R
R
S
R
R
Norfloxacin
S
R
R
R
S
R
R
Ciprofloxacin
-
-
R
R
S
-
R
Rifampicin
S
S
S
S
S
R
S
Fusidic acid
S
S
S
S
R
S
S
Fosfomycin
S
S
S
S
S
R
R
Vancomycin
S
S
S
S
S
S
R
Teicoplanin
S
S
S
S
S
L
R
Margaucin
3,125*
3,125
3,125
1,563
3,125
3,125
3,125
R resistant; S susceptible; L intermediate; - Not tested. These clinical multiresistant isolates have
* Minimal Inhibitory Concentration (MIC) of margaucin against Staphylococcus aureus clinical
diffusion test agar against Staphylococcus aureus CIP 76.25 and by analytical HPLC.
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
Purification Acetonitrile (10% v/v) and the adsorber resin Amberlite XAD-16 were added to the culture
multi-resistant strains (Table 2); nevertheless, it was also strongly active against Escherichia coli TolC, a Gram
broth, and agitated for 12 h at 4 °C. The XAD beads were separated from the culture broth, washed with water
negative strain deficient in a multidrug efflux transporter. This result suggests that resistance of Gram negative
and water/methanol (50/50) respectively, and eluted with methanol (100%). The eluate was concentrated by
bacteria to margaucin owes to the penetration barrier. No activity against Candida albicans, and no cytotoxic
Conclusion
evaporation under reduced pressure. The margaucin was finally purified by reverse phase HPLC using a
activity at doses up to 100 µg/ml in MCF7 (breast tumor cell) were observed. In vivo the molecule was not
This is the first report of a production of margaucin by a microorganism and of its antibacterial
toxic in mice at doses up to 100 mg/kg. Using a Staphylococcus aureus smith septicaemia model, we observed
activity. However the large scale synthesis of a molecule identical to margaucin has already
a protection of the mouse (OF1 female) at 100 mg/kg.
been described and is possible. This increases the potential interest of this molecule.
preparative C18 column (symmetryshield C18) and a linear gradient of H2O, 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.