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(12) Patent Application Publication (10) Pub. No.: US 2014/0017724 A1 LEONETT Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2014/0017724 A1 LEONETT Et Al US 20140017724A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0017724 A1 LEONETT et al. (43) Pub. Date: Jan. 16, 2014 (54) METHODS FOR ISOLATING BACTERIA (30) Foreign Application Priority Data (71) Applicants:CENTRE NATIONAL DE LA Jan. 19, 2009 (EP) .................................. O9305041.7 RECHERCHE SCIENTIFIOUE, Paris Cedex 16 (FR); DEINOVE, Paris (FR) Publication Classification (72) Inventors: JEAN-PAUL LEONETTI, Montpellier (51) Int. Cl. (FR); STEPHANIETEXIER, CI2P I/04 (2006.01) Montauban (FR) CI2R I/OI (2006.01) CI2P 23/00 (2006.01) (73) Assignees: CENTRE NATIONAL DE LA (52) U.S. Cl. RECHERCHE SCIENTIFIOUE, Paris CPC, C12P I/04 (2013.01): CI2P 23/00 (2013.01); Cedex 16 (FR); DEINOVE, Paris (FR) CI2R I/01 (2013.01) USPC ........... 435/67; 435/170; 435/71.3; 435/69.4: (21) Appl. No.: 14/030,504 435/101; 435/252.1 (57) ABSTRACT (22) Filed: Sep. 18, 2013 The present invention relates to compositions and methods to identify novel bacteria and metabolites derived therefrom. Related U.S. Application Data More specifically, the invention describes a novel method to isolate bacteria producing metabolites of interest from envi (63) Rsingit. E; ronmental samples. Particularly, the invention discloses a EP2010/05 o 51 3. O Jan 18, 201 p method to select rare antibiotic producing bacteria. The • Y-s invention can be used from any sample and allows the isola (60) Provisional application No. 61/145,606, filed on Jan. tion of bacteria having e.g., pharmaceutical or agrochemical 19, 2009. interest. Patent Application Publication Jan. 16, 2014 US 2014/0017724 A1 igure 1a Figure 1b US 2014/0017724 A1 Jan. 16, 2014 METHODS FOR ISOLATING BACTERIA soil is often only about 1% of the total number of cells present. Most of these bacteria are difficult to detect and isolate using 0001. The present invention relates to compositions and standard isolation techniques, either because they are rare in methods to identify novel bacteria and metabolites derived the environment, or because they are less adapted to environ therefrom. More specifically, the invention describes a novel mental conditions than other bacteria like Pseudomonas spp. method to isolate bacteria producing metabolites of interest or Bacillus spp. and are rapidly overgrown. Several isolation from environmental samples. Particularly, the invention dis techniques aiming to increase the diversity of the isolates closes a method to select novel antibiotic producing bacteria. have been published. For example, serial liquid dilution cul The invention can be used from any sample and allows the ture has been used successfully to improve cultivability isolation of bacteria having e.g., pharmaceutical or agro (Schoenborn et al., 2005) and to facilitate the isolation of chemical interest. bacteria from diverse environments. Yang et al., 2008, also described the use of microwave treatment to isolate rare acti BACKGROUND nomycetes. WO02/059351 concerns a method for enriching a 0002 For time immemorial, mankind has sourced sur microbial population from a natural environment using e.g., rounding organisms for therapeutic purposes, and most of the hot spring water. commercial antibiotics still have a natural origin. During the 0007. However, environmental microorganisms still rep last 10 years, in an effort to rationalize and to speed up resent a rich and unexploited resource of novel compounds antibacterial discovery processes, the industry has moved and activities, and there is a need in the art for improved or from natural products and a molecule-oriented discovery alternative methods to identify bacteria of interest. strategy to synthetic molecules and a target-oriented Strategy. 0003. After years of uniformization of antibiotic R&D SUMMARY OF THE INVENTION process, with an unprecedented high attrition rate, it is nec 0008. The present application provides a novel approach essary to return to natural resources. Indeed, the screening of to identify or isolate bacteria from environmental samples. natural product libraries usually yields a higher percentage of More particularly, the invention discloses an efficient and antibiotics hits than that of chemical libraries, and conse rapid method of identifying or isolating rare bacteria from quently provides a higher probability to obtain a therapeutic. environmental samples using a DNA damaging treatment. The first reason probably lies in the ecologic role of the 0009. An object of the present invention therefore resides antibiotics, which have been optimized through the course of in a method to identify or isolate a (secondary) metabolite evolution, to defend plants, animals and micro-organisms producing bacterium, the method comprising Subjecting a against other living organisms. Furthermore, natural products sample comprising uncharacterized bacteria to a cell are generally as lipophilic as combinatorial compounds, but destructing DNA damaging treatment and identifying or iso they have an unparalleled structural diversity and dispersion lating from said treated Sample a bacterium which produces in chemical space. This helps to find rare hydrophilic hits the (secondary) metabolite. In a preferred embodiment, the which can be optimized for in vivo applications. treatment is a repeated irradiation treatment. 0004. It is generally assumed that 20 to 30 percent of the 0010. In this respect, a particular object of this invention is bacteria isolated from environmental Sources such as Soil or a method to identify or isolate a metabolite-producing bacte water are antibiotics producers (Berdy, 2005). For obvious rium, the method comprising: reasons, the fitter and the most represented bacteria are the 0011 a) providing a sample comprising uncharacterized most frequently isolated. This explains why many antibiotics bacteria; produced by Bacillus or Pseudomonas have already been 0012 b) subjecting the sample to a repeated irradiation documented, and why it is more and more difficult to find new treatment, molecular entities produced by these genera. Other families 0013 c) identifying or isolating a living or growing bac ofunder-represented bacteria, more adapted to antibiotic pro terium from said treated Sample; and duction, due to a bigger genome, can be easily isolated with 0014 d) selecting a bacterium of step c) which produces adapted techniques. This is the case of actinomycetals, by far the metabolite. the best antibiotic producers (Berdy, 2005). They were exten 0015 Inaparticular embodiment, the treatment comprises sively studied by the pharmaceutical industry between 1950 a sequential UV treatment, e.g., a repetition of at least 2, and 1980, and it is now difficult to identify strains producing preferably 3 or more irradiations at essentially regular inter non-redundant molecules. vals. 0005. In the last decade, efforts have been devoted to iso 0016. In a particular embodiment, the treatment comprises lating rare bacteria in order to find new chemical entities of a repeated UV irradiation. pharmaceutical interest. Myxobacteria, for example, are 0017. According to another particular embodiment, the known since the 1940’s, but due to difficulties encountered invention relates to a method to identify or isolate a metabo for isolating them, they were under-represented in the collec lite-producing bacterium, the method comprising: tions. Thanks to an extensive collection campaign carried out 0018 a) providing a sample comprising uncharacterized by Reichenbach Hand collaborators, so far about 80 different bacteria; compounds and 450 structural variants produced by Myxo 0019 b) adding to the sample an antibiotic of the bleomy bacteria have been characterized (Reichenbach, 2001). Many cin family; of those compounds were new. Among them is the antine 0020 c) identifying or isolating a living or growing bac oplasic drug, epothilones, currently being evaluated in clini terium from said treated Sample; and cal trials. 0021 d) selecting a bacterium of step c) which produces 0006. It is generally recognized, however, that microbi the metabolite. ologists are unable to culture most soil microorganisms 0022. The preferred antibiotic of the bleomycin family is (Schoenborn et al., 2005). The number of cultivable cells in bleocin. US 2014/0017724 A1 Jan. 16, 2014 0023 The metabolite may be any pharmaceutical product, LEGEND TO THE FIGURES Such as antibiotics, bacteriostatic compounds, anti-metabo lite agents, chemotherapeutic compounds, anti-fungal agents, 0038 FIG. 1: Bacterial mat obtained after: a) 1 exposure to anti-viral compounds, cytokine-activity compounds or cell UV: 0, 4 and 17 m.J/cm and b) repeated exposures to UV growth factors. treatment of 4 m.J/cm. Dark colonies are D. radiopugnans. 0024. Another object of this invention is a method to iden The other bacteria belong to soil community. tify or isolate a bacterium which produces an antibiotic or a bacteriostatic-compound, the method comprising: DETAILED DESCRIPTION OF THE INVENTION 0.025 a) providing a sample comprising uncharacterized 0039. The present application describes a novel method to bacteria; identify or isolate bacteria from samples. More particularly, 0026 b) subjecting the sample to a repeated irradiation the invention discloses an efficient and rapid method of iden treatment, preferably a repeated UV treatment; tifying or isolating rare bacteria from environmental samples 0027 c) identifying or isolating living or growing bacteria using a cell destructing DNA damaging treatment. The inven from said treated sample; and tion further relates to methods
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