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 of producing pharmaceutical 0028 d) exposing identified or isolated bacteria of step c), compounds using Such bacteria. or an extract thereof, to a reference bacterial strain and iden tifying or isolating a bacterium which exhibits an antibiotic or Isolation and Culture of Bacteria bacteriostatic activity. 0040. Resistance of bacteria to UV or radiation has been 0029. In a preferred embodiment, the methods of the extensively studied to understand how bacteria can survive in present invention comprise a further step of isolating or puri Such aggressive environment (Makarova et al., 2001). Rainey fying the metabolite produced by said bacterium. eta.l., 2005, showed that Geodermatophylus spp. and Deino 0030. Furthermore, the methods of the invention option coccus spp. are among the most radiation-resistant bacteria ally comprise a further step of modifying, either genetically, collected from the Sonoran desert soils. Other bacteria were biologically or chemically, the identified or isolated bacteria also isolated namely, Deinococcus, Hymenobacter, Kine or their DNA by any technical process known per se by a ococcus, Kocuria, and Methylobacterium. skilled person, said modification aiming to improve e.g., the 0041. The present invention now proposes the use of a cell viability, growth or function of the said bacteria, e.g., in order destructing DNA damaging treatment to isolate novel (under to improve the antibiotic activity or production. This represented) bacteria producing metabolites. The present includes, without limitation, cell fusion, accelerated evolu invention indeed shows that Such a treatment, which would tion, DNA shuffling technologies, insertion of eukaryote, normally cause Substantial cell death, unexpectedly allows prokaryote or synthetic nucleic acid (e.g., DNA) from another the selection of under-represented bacteria having remark strain, or any genetic engineering technology. Said modifica able properties of producing valuable metabolites. tion step can be carried out on the isolated bacteria, or at any 0042. The invention now shows for the first time that, after earlier stage of the process, e.g., on the sample of step a), for a cell destructing DNA damaging treatment, it is possible to instance. isolate a high number of bacteria able to produce valuable 0031. Another object of this invention resides in a wild secondary metabolites. type or modified bacterium obtained by a method as disclosed 0043. An object of the present invention therefore resides above, or an extract thereof. in a method to identify or isolate a metabolite producing 0032. A further object of this invention is a method of bacterium, the method comprising Subjecting a sample com producing a metabolite, particularly a pharmaceutical com prising uncharacterized bacteria to a cell destructing DNA pound, the method comprising (i) identifying or isolating a damaging treatment and identifying or isolating from said bacterium which produces said metabolite using the method treated sample a bacterium which produces the metabolite. as defined above and (ii) producing said antibiotic. 0044. In a particular embodiment, the method comprises: 0033. A further object of this invention is a method of 0045 a) providing a sample comprising uncharacterized identifying, isolating or producing a pharmaceutical com bacteria; pound (such as an antibiotic), using a wild type or modified 0046 b) subjecting the sample to a cell destructing DNA Deinococcus bacterial strain. damaging treatment; 0034. A further object of this invention resides in the use of 0047 c) identifying or isolating a living or growing bac a wild type or modified Deinococcus bacterium to produce a terium from said treated Sample; and pharmaceutical compound (Such as an antibiotic). 0048 d) selecting a bacterium of step c) producing the 0035 A further object of this invention is an antibiotic metabolite. derived from a wild type or modified Deinococcus bacteria. 0049. The method can be implemented with various 0036. A further object of this invention is a method of samples comprising uncharacterized bacteria, particularly producing a recombinant host cell, the method comprising with samples which are or derive from an environmental identifying or isolating a bacterium according to the method sample. Within the context of this invention, environmental as defined above and cloning one or several genes (or corre samples include any sample containing (a plurality of) sponding synthetic or recombinant nucleic acids) from said uncharacterized (micro)organisms, particularly uncultivated bacterium in another host cell, thereby producing a recombi microorganisms (e.g., microorganisms that have not been nant host cell. purposely cultured and expanded in isolated form). The 0037. The methods of the present invention can be used sample may be obtained or derived from natural environ with various samples, such as environmental samples, and ments or from artificial or specifically created environments. has been used Successfully to isolate novel bacteria having 0050. As indicated, the sample may be any environmental advantageous pharmaceutical and/or agro-chemical proper sample, such as those obtained or derived from soil, water, ties. Vegetal extract, biological material, sediments, peatlands, US 2014/0017724 A1 Jan. 16, 2014

industrial effluents or sites, mineral extracts, sand, and the sp. Sphingobacterium sp. Cellulosimicrobium sp. Tepidimo like. The sample may be collected from various regions or nas sp. Truepera sp., Porphyrobacter sp., Novosphingobium conditions, such as but not limited to tropical regions, Volca sp., Exiguobacterium sp., Nocardia sp Arthrobacter sp., nic regions, forests, farms, industrial areas, etc. The sample Rhodococcus sp., Microbacterium sp., Kineococcus sp., and usually contains various species of (uncharacterized, uncul Williamsia sp. tivated) microorganisms, such as terrestrial microorganisms, 0058. The DNA damaging treatment may comprise sub marine microorganisms, freshwater microorganisms, symbi jecting the sample to irradiation(s) and/or to one or several otic microorganisms, etc. Species of Such environmental genotoxic agents. The treatment is conducted under condi microorganisms include bacteria, algae, fungi, yeasts, tions and/or for a period of time sufficient to induce substan moulds, viruses, etc. The microorganisms may include extre tial cell death in the microorganisms present in the sample. mophile organisms, such as e.g., thermophiles. The sample 0059. In a preferred embodiment, the DNA damaging typically comprises various species of Such (uncultivated) treatment comprises subjecting the sample to one or several microorganisms, as well as various amounts thereof. Further irradiations. A preferred treatment comprises subjecting the more, the sample may contain, in addition, known and/or sample (i.e., microorganisms in the sample) to a repeated cultivable microorganisms (e.g., prokaryotic or eukaryotic). (e.g., sequential) irradiation treatment. 0051. It should be understood that the present invention is 0060 Irradiation may be selected from UV, gamma and/or not limited to any specific type of sample or environmental X ray irradiation, either alone or in combinations, most pref microorganism, but can be implemented using any sample erably UV irradiation(s). Irradiation treatment typically com comprising uncultivated microorganisms. prises Subjecting the microorganisms to one or several 0052. In a preferred embodiment, the sample is or derives sequential irradiations (e.g., from 1 to 5), which may be of the from soil, water, hot springs, marine environment, mud, same or different nature, preferably of the same nature. wood, Stone, moss, vegetal extract, lichen, biological mate Repeated irradiation treatments are typically carried out at an rial, sediment, biofilm, industrial effluents, gas, sand, oil, interval of between 1 and 8 hours, preferably 3 to 5 hours, and sewage, or animal or human dejection. more preferably of about 4 hours. 0053 For use in the present invention, the sample may be 0061 A particularly preferred treatment comprises sub wet, Soluble, dry, in the form of a suspension, paste, etc. jecting the sample to a cell destructing UV irradiation. The Furthermore, prior to stepb) of the method, the sample may invention indeed shows that Such a treatmentallows to isolate be treated to improve the process, for instance to enrich for with high efficacy from environmental (e.g., Soil or water) microorganisms, e.g., such as through filtration, washings, samples, under-represented bacteria species producing (sec concentration, dilution, Steering, drying, etc. ondary) metabolites (such as antibiotics). Cell destructing 0054. In a particular embodiment, the sample is in the UV treatments are typically of between 0.5 and 400 m.J/cm2, form of a filtered suspension. More particularly, the sample more preferably of between 1 and 200 m.J/cm2, typically may be sterile-filtered and/or placed in sterile water, prior to between 1 and 100 m.J/cm2, applied for a period of time of treatment step b). about 5" to 5". A preferred UV treatment is 4 mJ/cm2 for 30 0055 Step b) of the process comprises subjecting the seconds. sample (i.e., microorganisms contained in the sample) to a 0062. In a specific embodiment, the cell destructing DNA cell destructing DNA damaging treatment. damaging treatment comprises Subjecting the sample to at 0056. The cell destructing DNA damaging treatment des least 2, preferably at least3 UV treatments of between 0.5 and ignates a treatment that causes Substantial cell death in the 400 m.J/cm2 each, preferably of about 4 m.J/cm2 each, carried sample, as opposed to mere mutagenic treatments which out at an interval of between 1 and 8 hours, preferably 3 to 5 introduce DNA modifications. In particular, the cell destruct hours, and more preferably of about 4 hours. ing DNA damaging treatment is a treatment that is sufficient 0063. In an alternative method, the cell destructing DNA to cause 90% cell death, or more, in a culture of E. coli damaging treatment comprises contacting the sample with a bacteria. Even more preferably, the cell destructing DNA genotoxic agent, such as a solvent, mitomycin, a bleomycin damaging treatment is a treatment that is sufficient to reduce antibiotic, or HO. It should be understood that genotoxic by at least 2 log the bacterial titer in a culture of E. coli. agents may also be used in combination with irradiation. In a Surprisingly, the invention shows that such a treatment, which particular embodiment, the treatment step b) comprises add would normally be lethal to most cell populations, allows the ing to the sample an effective amount of bleocin, a bleomycin efficient and rapid isolation of novel microorganisms from family antibiotic. As illustrated in the examples, the addition various types of samples, which microorganisms produce of bleocin causes substantial bacterial cell death while allow valuable (secondary) metabolites. This result is particularly ing rare, metabolite-producing bacteria to grow. Surprising since Subjecting microorganisms to Such cell 0064. During the treatment phase, the sample is preferably destructing DNA damaging treatment would have been placed in a Suitable culture medium such as, without limita expected to prevent isolation of living microorganisms. Sur tion, PGY (Bacto-peptone 10 g/L. Yeast extract 5 g/L glucose prisingly, the invention allows the selection of rare microor 20 g/L) or LB (Bacto-tryptone 10 g/L, Yeast extract 2.5g/L, ganisms from the sample, especially microorganisms having Sodium chloride 10 g/L). It should be understood that other the ability to either reassemble their genome or to resist DNA suitable culture media are known to the skilled person damage. (Buchanan et al., 1974. Difico, 1995)) or may be prepared by 0057 The invention thus discloses, for the first time, the the skilled person from Such known media. use of a cell destructing DNA damaging treatment to select, 0065 Treatment stepb) is typically performed in a solidor with a high efficacy, under-represented bacteria producing semi-solid culture medium, Such as in the presence of a gel valuable (secondary) metabolites. As illustrated in the (e.g., agar). A most preferred treatment medium comprises an examples, the invention allowed the isolation of novel bacte agar culture medium, Such as a Softagar culture medium. In a rial strains of Deinococcus sp., Bacillus sp. Methylobacterium particular embodiment, a TGY agar medium is used to grow US 2014/0017724 A1 Jan. 16, 2014

the bacteria. However, different solid media containing a Deinococcus, Truepera, Porphyrobacter sp., Novosphingo carbon Source, a nitrogen source and mineral salts can be used bium sp., Exiguobacterium sp., Chronobacterium sp. Arthro as well. Serial dilution techniques can also be used according bacter sp., Rhodococcus sp., Microbacterium sp. Kineococcus to Schoenborn et al. 2004. sp, or Williamsia sp have been isolated from different envi 0066. In step c), living or growing bacteria are identified or ronmental samples, including water, soil, animal dejection, isolated from the treated Sample. Living or growing bacteria wood, etc. may be identified by different means known perse in the art. 0075. The selection of bacteria which produce antibiotics In a particular embodiment, colonies which form in the cul can be performed using different techniques known perse in ture media are identified. The living or growing bacteria can the art (Singh, 2008, Janssen, 2002, Hamaki, 2005, Schoen be isolated and placed in fresh medium for further culture or born, L. et al. 2005). In a particular embodiment, the tested expansion. bacteria (or an extract thereof) are placed in contact with one 0067. As mentioned above, the method of this invention or several reference bacterial strains, and the ability of the test preferably comprises a step d) of selecting one or several bacteria to inhibit the growth of or to kill the reference strains bacteria, from the identified or isolated living or growing is measured, as an indication of the antibiotic activity. bacteria, which produce a particular metabolite. In this Examples of reference strains include, without limitation, E. regard, it should be noted that steps c) and d) can be per coli, Staphylococcus aureus, or Candida tropicalis (Lorian, formed sequentially, in any order, or simultaneously. For 1996). instance, the bacteria in the sample may be placed under 0076. In this regard, the invention relates to a method to conditions suitable to select the desired activity in step b) or identify or isolate bacteria producing an antibiotic or bacte c), so that growing or living bacteria identified or isolated in riostatic agent, the method comprising: step c) exhibit the desired activity. Alternatively, the bacteria 0077 a) providing a sample comprising uncharacterized identified or isolated in step c) may be placed under condi bacteria; tions suitable to select the desired activity in step d) only, so 0078 b) subjecting the sample to a cell destructing DNA that growing or living bacteria are first identified or isolated in damaging treatment, preferably a repeated irradiation treat step c) and then selected for the desired activity. ment, e.g., an UV treatment; 0068. The metabolite may be any compound having phar 0079 c) identifying or isolating living or growing bacteria maceutical and/or agro-chemical interest. In a particular from said treated Sample; and embodiment, the metabolite is a pharmaceutical compound 0080 d) exposing identified or isolated bacteria of step c), (for use inhuman or veterinary medicine), preferably selected or an extract thereof, to a reference bacterial strain and iden from antibiotics, bacteriostatic compounds, anti-metabolite, tifying or isolating bacteria which exhibit an antibiotic or chemotherapeutic compounds, anti-parasitic agents, anti bacteriostatic activity. fungal agents, anti-viral compounds, cytokine-activity com I0081. The method may further comprise a step of isolating pounds or cell-growth factors. or purifying the antibiotic produced by said bacteria. 0069. The metabolite may also have utility e.g., in cosmet I0082. A further object of this invention is a method to ics or agriculture, such as pigments, insecticides, pesticides, identify or isolate anti-metabolite or chemotherapeutic or chemical-degrading compounds, etc. anti-fungal or cytokine activity or cell-growth factor produc 0070 The selection or identification or bacteria producing ing bacteria, the method comprising: the selected metabolite can be made according to techniques 0083 a) providing a sample comprising uncharacterized known perse in the art. In a particular embodiment, step d) bacteria; comprises exposing identified or isolated bacteria of step c), I0084 b) subjecting the sample to a cell destructing DNA or an extract thereof, to one or several indicator cells and damaging treatment, preferably a repeated irradiation treat selecting a bacterium which affect the viability, growth, ment, e.g., an UV treatment; metabolism, mobility, RNA expression, protein expression, I0085 c) identifying or isolating living or growing bacteria protein secretion or virus production of at least one of said from said treated Sample; and indicator cells. I0086 d) exposing identified or isolated bacteria of step c), 0071. In the case of antibiotic or antibiostatic agents, the or an extract thereof, to one or several reference eukaryotic indicator cells are typically reference bacterial Strains and test cell types and identifying selected bacteria which affect the bacteria which inhibit the growth or kill said reference strains viability, growth, metabolism, mobility, RNA expression, are selected. protein expression, or protein secretion of some of the 0072. In the case of, e.g., anti-viral compounds, the indi eukaryotic cell types. cator cells are typically virus-producing cells and bacteria I0087. The method may further comprise a step of isolating which affect the production of virus or viability of virus or purifying the anti-metabolite or chemotherapeutic or anti infected cells are selected. fungal or cytokine activity or cell-growth factor produced by 0073. In this regard, in a particular embodiment, the said bacteria. method of the invention further comprises a step of isolating I0088 A further object of the invention resides in a method or purifying the metabolite produced by said bacteria. to identify or isolate anti-viral compound producing bacteria, 0074. In a preferred embodiment, the selected activity is the method comprising: the production of an antibiotic or bacteriostatic activity. The I0089 a) providing a sample comprising uncharacterized present invention shows that antibiotic-producing bacteria bacteria; can be isolated with high efficiency from environmental 0090 b) subjecting the sample to a cell destructing DNA samples treated according to the present method. More par damaging treatment, preferably a repeated irradiation treat ticularly, as illustrated in the examples, antibiotic-producing ment, e.g., an UV treatment; bacteria of the genus Bacillus, Methylobacterium, Sphingo 0091 c) identifying or isolating living or growing bacteria bacterium, Cellulosimicrobium, Tepidimonas, Nocardia, from said treated Sample; and US 2014/0017724 A1 Jan. 16, 2014

0092. d) exposing identified or isolated bacteria of step c), larly, the bacteria are maintained at the selected pH during or an extract thereof, to one or several virus-producing cell step b), c) and/or d); and/or during an additional step e), in types to select bacteria which affect the production of virus or order to identify or isolate bacteria which are viable or can be viability of virus-infected cells. grown at the desired pH. 0093. The method may further comprise a step of isolating 0105. In a further particular embodiment of the present or purifying the anti-viral agent produced by said bacteria. invention, the bacteria are cultured under particular oxygen 0094. The methods of the present invention may also ation conditions in order to identify or isolate bacteria which include a step of determining the genus/species of the iden are viable or can be grown in aerobic and/or anaerobic con tified or isolated bacteria. In this respect, the invention is ditions. More particularly, the bacteria are maintained under particularly Suited for identifying or isolating Deinococcus the selected oxygenation conditions during stepb), c) and/or bacteria which produce a selected metabolite. d); and/or during an additional step e), in order to identify or 0095 Accordingly, in a particular embodiment, the inven isolate bacteria which are viable or can be grown at the tion relates to a method for identifying or isolating a bacte desired conditions. rium which exhibit a selected activity (e.g. which produces a 0106. In a further particular embodiment of the present metabolite of interest), the method comprising: invention, the bacteria are cultured in a particular culture 0.096 a) providing a sample comprising uncharacterized medium in order to identify or isolate bacteria which are bacteria; viable or can be grown in the presence of a selected carbon 0097 b) subjecting the sample to a cell destructing DNA Source. More particularly, the bacteria are maintained under damaging treatment, preferably a repeated irradiation treat the medium during step b), c) and/or d); and/or during an ment, additional stepe), in order to identify or isolate bacteria which 0098 c) identifying or isolating living or growing Deino are viable or can be grown using the desired carbon Source. coccus bacteria from said treated Sample; and 0107. It should be understood that the above characteris 0099 d) selecting Deinococcus bacteria of step c) which tics can be selected individually or in any combinations. For exhibit the selected activity. instance, the method can be used to identify bacteria which 0100. The bacteria can be characterized and classified e.g., are viable or can be grown at a desired temperature and according to Hirsch, 2004 and Kampfer, 2008. The physi salinity, or at a desired temperature and pH, or at a desired ological characterization includes for instance the determina temperature, pH and oxygenation condition. Furthermore, tion of the fatty acid, respiratory quinones, polar lipids and the methods of this invention may comprise a further step of polyamines patterns, a metabolic profiling of different modifying, e.g., either biologically, genetically and/or chemi sources of carbon and/or the determination of the 16S rRNA cally, the identified or isolated bacteria, or their DNA, by any gene Sequences. process known perse in the art, said modification aiming e.g., 0101. Furthermore, the methods of this invention can to improve the viability, growth or functions of the said bac comprise one or several additional steps of selecting bacteria terium, e.g., in order to improve the antibiotic activity. Such having particular properties. More particularly, in a preferred modification step includes, without limitation, cell fusion, embodiment, the method further comprises one or several accelerated evolution, DNA shuffling, mutagenesis, insertion steps of selecting bacteria which are viable or grow under of eukaryote, prokaryote or synthetic nucleic acid (e.g., selected culture conditions, such as media, temperature, pH, DNA) from another strain, or any genetic engineering tech salinity, nutrients, oxygenation or carbon source. For this nology. The modification may also include a step of introduc purpose, the sample or bacteria can be placed under appro ing a marker gene (e.g., kanamycin resistance) in the bacte priate selection conditions during any one of steps b), c) ord), rium. or during a prior or Subsequent step, and the resulting prop 0108. Many (marketed) antibacterial drugs are semisyn erty is selected for during any of these steps. thetic analogs of natural products, and are obtained from 0102. In a particular aspect of the present invention, the modifications of initial fermentation products. This is the case bacteria are cultured under particular temperature conditions for Ketolides, the B-lactam group of antibiotics. Semisynthe in order to identify or isolate bacteria which are viable or can sis or even manipulation of the enzymes involved in the be grown in a temperature range from approximately 4 to 70° antibiotic biosynthesis can therefore be used to improve the C. More particularly, the bacteria are maintained at the initial hit (Von Nusssbaum, 2006, Marsden, 1998). selected temperature during step b), c) and/or d); and/or dur 0109 Accordingly, in a particular embodiment, the inven ing an additional step e), in order to identify or isolate bacteria tion resides in a method of producing a bacterium having a which are viable or can be grown at the desired temperature. selected activity, the method comprising isolating a bacte 0103) In another particular aspect of the present invention, rium according to any of the above described methods and the bacteria are cultured under particular saline conditions in modifying said bacterium e.g., by genetic, biologic or chemi order to identify or isolate bacteria which are viable or can be cal treatment to improve said activity. grown under concentration conditions of NaCl or equivalent 0110. As illustrated in the examples, the method of the salts possibly reaching around 5% weight/volume. More par present invention has allowed the efficient and rapid isolation ticularly, the bacteria are maintained at the selected Salinity of UV-resistant bacteria which produce secondary metabo during step b), c) and/or d), and/or during an additional step lites (e.g., antibiotics). These bacteria were unknown before e), in order to identify or isolate bacteria which are viable or and can extend the diversity and spectrum of antibiotic activ can be grown at the desired salinity. ity (or other secondary metabolites) for therapeutic or other 0104. In a further particular and preferred embodiment of industrial purposes. the present invention, the bacteria are cultured under particu 0111. A further aspect of this invention therefore also lar pH conditions in order to identify or isolate bacteria which resides in a bacterium obtainable by a method as disclosed are viable or can be grown in a pH interval between approxi above, or an extract thereof. The extract may be any prepara mately 3 and 9.5, preferably between 4 and 8. More particu tion derived from a cell culture, such as a Supernatant, a US 2014/0017724 A1 Jan. 16, 2014 lysate, a cell membrane, or enriched/purified preparations therapeutic compounds, antioxidants, anti-inflammatory, derived therefrom, preferably containing a secondary polysaccharides, anti-parasitic agents, anti-fungal agents, metabolite. anti-viral compounds, cytokine-activity compounds, cell 0112 The bacterium may belong to different genus, such growth factors, hormones, anti-depressives, anti-migraine, as, e.g., Bacillus, Methylobacterium, Sphingobacterium, Cel anti-asthma, contraceptives, anti-diabetics, psychotropic, lulosimicrobium, Tepidimonas, Nocardia, Deinococcus, anti-arythmics, enzyme-inhibitors, or adjuvants. Truepera, Porphyrobacter sp., Novosphingobium sp., Exig 0.120. The term “drug also includes, generally, any agent uobacterium sp., Chronobacterium sp. Arthrobacter sp. that improves or modifies Health or well-being, alicaments, Rhodococcus sp., Microbacterium sp. Kineococcus sp., or Wil probiotics, etc. liamsia sp. I0121 Probiotics designate live microorganisms that, 0113. The invention also relates to a method of producing when administered inadequate amounts, confer a health ben a recombinant host cell, the method comprising identifying or efit on the host (Sanders M. E. 2008 Probiotics: definition, isolating or producing a bacterium according to any one of the sources, selection, and uses. Clin Infect Dis. February 1:46). methods as defined above and cloning genes (or gene clusters Probiotics may facilitate a return to normal status after a or operons, or corresponding synthetic or recombinant perturbation of the microbiota (e.g., because of the use of nucleic acids) from said bacterium into another host cell, antibiotics or illness), they may aid in establishing the best thereby producing a recombinant host cell. The host cell may balance of microorganisms or to improve the balance of the be a prokaryotic or eukaryotic host cell, preferably a prokary intestinal microbiota, (Balcazaret al., 2006. The role of pro otic host cell. Cloning can be made according to techniques biotics in aquaculture. Veterinary Microbiology 114, 173 known perse in the art, such as using cloning vectors (e.g., 186). Others benefits of probiotics include (i) competitive plasmids, phages, bacterial chromosomes, etc.). In a pre exclusion of pathogenic bacteria, (ii) source of nutrients and ferred embodiment, genes or gene clusters encoding all or enzymatic contribution to digestion; (iii) direct uptake of part of a biosynthetic pathway (e.g., genes encoding enzymes dissolved organic material mediated by the bacteria or (iv) involved in such pathways) are isolated from the identified enhancement of the immune response against pathogenic bacterium, cloned into appropriate vectors, and inserted into microorganisms. the selected host cell, to build new biosynthetic pathways. 0.122 For use as probiotics for human food, Deinococcus sp. may be formulated in e.g., yogurt or fermented milk or Drug Microbial Production drinks, typically at a dosage of between about 110° and about 0114. The invention also relates to a method of producing 1:10 CFU per day or per dose. a pharmaceutical agent (Such as an antibiotic), the method I0123. The pharmaceutical agent or drug may be a small comprising identifying or isolating or producing bacterium chemical compound, a peptide, a protein, a lipid, a polysac according to any one of the above methods, wherein said charide, a nucleic acid, etc. bacterium produces the pharmaceutical agent, and producing 0.124. In this regard, aparticular object of the invention lies said antibiotic. Production of the pharmaceutical agent may in a method for producing a drug, comprising (i) culturing, in comprise culturing the bacterium (or progeny or derivatives a suitable medium, a bacterium which produces said drug or thereof) under Suitable conditions are collecting or purifying an intermediate (or precursor) thereof, (ii) collecting or puri the agent. Production of the agent may also comprise artificial fying the drug or intermediate from the culture, and (iii) when synthesis (e.g., chemical, recombinant, enzymatic, etc.). an intermediate is produced in step (i), converting said inter 0115 A further object of this invention is a method of mediate into said drug, wherein said bacterium is a Deino identifying, isolating or producing a pharmaceutical agent coccus or a related bacterium. (such as an antibiotic), using a wild type Deinococcus bacte rial strain or a related bacterium. 0.125 Within the context of the present invention, an 0116. A further object of this invention is a method of “intermediate' of a drug refers to any substance formed in the identifying, isolating or producing a pharmaceutical agent course of the reactions that lead to said drug. Such interme (such as an antibiotic), using a modified Deinococcus bacte diate may exhibit a pharmacological activity. According to rial Strain. the invention, the term “intermediate' encompasses the pro 0117. A further object of this invention resides in the use of drugs. For instance, a prodrug may designate an inactive a Deinococcus or related bacterium to produce a pharmaceu intermediate whose metabolism may lead to an active tical agent (Such as an antibiotic). metabolite, i.e. the drug. 0118 Within the context of the present invention, the 0.126 Another particular object of the invention is an terms “pharmaceutical agent” and "drug are used inter improved method for the production of a drug from a cultured changeably and designate any Substance (Suitable for use in a microbial cell, the improvement consisting in the use, as said pharmaceutical or veterinary product) which exhibits a phar microbial cell, of a Deinococcus or related bacterium. macological activity and/or which have a direct effect in the I0127. A further object of the invention relates to a method cure, mitigation, treatment or prevention of a disease, and/or for producing a drug by transformation of a precursor thereof, which have direct effect in restoring, correcting or modifying said method comprising exposing the precursor to a catalyst a physiological function in a human being or in a non-human and collecting the drug resulting from the transformation, animal. wherein the catalyst is a Deinococcus or a related bacterium, 0119 The term “drug includes, for instance, any chemi or an extract thereof. cal Substance used in the treatment, cure, prevention, or diag I0128. Upon production, or isolation or collection, the drug nosis of a disease or used to otherwise enhance physical or may be used as such or may be further chemically modified, mental well-being in mammals, particularly inhuman beings. as appropriate. Also, the drug may be formulated in any Specific examples of drugs include, without limitation, anti appropriate composition using Suitable excipients, diluents or biotics, bacteriostatic compounds, anti-metabolite, chemo carriers. US 2014/0017724 A1 Jan. 16, 2014

0129. A further object of this invention is an antibiotic 0.138. Further aspects and advantages of the instant inven derived from a Deinococcus bacterium. tion will be disclosed in the following examples, which 0130. A further object of this invention is an antioxidant should be regarded as illustrative and not limiting. All refer derived from a Deinococcus bacterium. ences (patent applications, patents, scientific publications) 0131) A further object of the invention is a pharmaceutical cited in this application are incorporated therein by reference. composition comprising a Deinococcus bacterium. 0132 A further object of the invention is a food additive composition comprising a Deinococcus bacterium. EXAMPLES 0133. A further object of the invention is a composition comprising a Deinococcus bacterium as an intestinal flora Example 1 modifier. 0134. The invention discloses the ability of Deinococcus bacteria to naturally produce drugs of high value or interest, Determination of Improved Conditions for Selecting or precursors thereof. The invention may therefore use such Metabolite-Producing Bacterium bacteria without further modification, or use improved or modified bacteria with increased production levels or quality. 0.139. In a preliminary step, 5 strains were tested for their In particular, the bacteria may be selected by culture inappro resistance to UV. These strains were E. coli, Deinococcus priate conditions and medium to selected best clones, or radiopugnans, Deinococcus radiodurans, Deinococcus sp. cultured under selection/evolution pressure to increase the and Deinococcus geothermalis. performance, and/or genetically modified. 0140 Stationary phase cultures of the 5 tested strains were 0135 Specific examples of Deinococcus-related bacteria include Microbacterium sp., Promicromonospora sp., and successively diluted from 10° to 10. All dilution were spot Chronobacterium haemolyticum. ted (5 uL) on agar rich media TGY (Trypton 10 g/l. Glucose 0136. Also, the present invention can be used to select 2.5 g/L and Yeast extract 5 g/L) and different UV-treatments bacteria and to generate metagenomic libraries. Typically were applied: 0 m.J/cm, 4 m/cm, 17 mJ/cm, 42 mJ/cm genomic DNA from Such bacteria is extracted and large frag and 167 m.J/cm with a BioLink crosslinker (Vilbert Lour ments of DNA are (directly) cloned in a shuttle vector to make mat). a metagenomic library representative of said bacteria. This metagenomic library can be transferred into different host 0141 Table 1 below presents the bacterial density after bacteria to identify secondary metabolite producing strains. different UV treatments. TABLE 1

Strains O mJ/cm2 4 mil/cm 17 ml/cm 42 mJ/cm2 167 m.J/cm? D. radiopugnans (CFU/ml) 4.00E+09 1.00E+09 1.6OE+08 6.00E+05 0.00E+00 D. radiodurans (CFU/ml) 2.OOE--O7 6.OOE-06 2.OOE-06 2.OOE--O3 OOOE--OO E. coli (CFU/ml) 3.OOE--O7 2.OOE-04 OOOE--OO O.OOE--OO O.OOE--OO D. geothermalis (CFU/ml) 1.2OE-09 6.OOE-08 8.OOE-05 O.OOE--OO O.OOE-00 Deinococcus sp. (CFU/ml) 8.OOE-08 2.2OE-08 6.OOE-06 4.OOE-03 O.OOE--OO

As an example, enzymes responsible for polyketide produc I0142. As can be seen, a treatment of 4 m.J/cm reduces by tion can be screened according to Ginolhac, 2007. 3 log the bacterial titer in a culture of E. coli. A treatment of 0.137 Furthermore, the invention also relates to improved 17 mJ/cm also discriminates between Ecoli and Deinococ industrial methods of using microorganisms. Indeed, the cus bacteria. invention also proposes to include one or several cell destruct 0143. In a second step, a Soil suspension was subjected to ing DNA damaging treatments into industrial processes in different irradiation treatments based on the results of table 1. order to eliminate potentially contaminating bacteria. Indeed, The Soil suspension was prepared as follows: 5g of wet soil since bacteria of the present invention are able to grow and was diluted in 20 mL distilled water. After Vortex and soni Survive such cell-destructing treatments, the latter may be cation (1 minute), Soil Supernatant was collected and mixed used, e.g., in any industrial fermentation or culture device or with a 350 uL. radiopugnans culture in stationary growth installation, to avoid contamination. In this respect, a particu phase. Analiquot (50LL) was spread on TGYagar containing lar object of this invention also resides in a process of using a an antifungal (cycloheximide, 100 g/mL), and different bacterium obtainable by a method as described above in a repeated sequential irradiation treatments were tested: 1 treat fermentation or continuous culture, wherein the process com ment, 2 treatments and 3 treatments. 4 h-intervals was prises a step of irradiation of the bacterium prior to or follow respected between each UV treatment. ing initiation of the fermentation or of the continuous culture, 014.4 FIG. 1a shows the results obtained after one expo to limit contamination by other bacteria or cells. A further sure at 0 m.J/cm, 4 m.J/cm and 17 m.J/cm. FIG.1b shows the object of this invention is a process of using a Deinococcus results obtained after 1, 2 and 3 exposures to 4 m/cm. bacterium in a fermentation or continuous culture, wherein (0145 As can be seen, the best tested conditions are 3 the process comprises a step of irradiation of the bacterium exposures to UV irradiations of 4 mJ/cm each. Such condi prior to or following initiation of the fermentation or of the tions provide the highest amount of growing Deinococcus continuous culture, to limit contamination by other bacteria bacteria from the treated soil sample. A substantial amount of or cells. Irradiation may be performed repeatedly, typically Deinococcus bacteria is also obtained after one exposure of under the conditions described above. the sample to UV irradiation of 17 m.J/cm. US 2014/0017724 A1 Jan. 16, 2014

Example 2 0154 Examples of colonies identified are listed in Table 2.

Isolation of UV-Resistant Bacteria from Water TABLE 2 Samples Isolation 0146 Water samples were concentrated by filtration overa Biotope Genus to C. 0.22 um nitrocellulose filter (Millipore, France), then placed Pebble Bacilius 30° C. in suspension in 10 ml of sterile water. The filtered solution is Stones Methyliobacterium 30° C. then Sonicated for approximately 60 seconds to resuspend the Animal dejection Sphingobacterium 30° C. Stones Cellulosimicrobium 30° C. bacteria. Water Tepidimonas 45° C. 0147 Following sonication, between 150 ul and 2 ml of Water Deinococcus 45° C. the Suspensions are spread on a solid PGY-agar enriched Pebble Deinococcus 30° C. culture medium sterilized by autoclaving (20 minutes at 120° Muck, pebble, soil Chronobacterium 30° C. C.) containing glucose (Sigma-Aldrich, France) 1 g/l, pep tone (Fluka, France) 10 g/l, and yeast extract (Fluka, France) 5 g/l. The seeded culture then undergo 3 UV treatments of 4 Example 5 mJ/cm each using a BLX-E254 biolink (Vilber-Lourmat, France), carried out at an interval of 4 hours. After incubation Isolation of Bacteria from Bleocin-Treated Samples at 30 to 50° C. for 3 to 4 days, the viable colonies of interest O155 Soil samples containing Deinococcus radiopugn were visible. ans strain DRHao were placed in suspension under conditions 0148 Examples of colonies identified are listed in Table 2 similar to that of example 2. In place of irradiation, the Sus below. pensions were maintained in the absence (control) or pres ence of 50 lug/ml or 100 ug/ml of bleocin. Example 3 0156. In the absence of bleocin, no growing pink colonies (Deinococcus) are observed, but only indigenous microflora. Isolation of UV-Resistant Bacteria from Wood and In the presence of 50 g/ml bleocin, indigenous microflora Pebble Samples decreases drastically, and pink colonies are present. The same phenomenon is observed at 100 g/ml of bleocin. 0149 Wood and pebble samples were immersed in sterile water then vortexed and sonicated for approximately 60 sec Example 6 onds. 0150. Following sonication, between 150 ul and 2 ml of Identification of Antibiotic Producing Bacteria the Suspensions are spread on a solid PGY-agar enriched culture medium sterilized by autoclaving (20 minutes at 120° 0157. The UV resistant strains as disclosed in Table 2 were C.) containing glucose (Sigma-Aldrich, France) 1 g/l, pep tested for their ability to inhibit the growth of several refer tone (Fluka, France) 10 g/l, and yeast extract (Fluka, France) ence strains of bacteria. Briefly, fermentation was carried out 5 g/l. The seeded culture media then undergo 3 UV treatments in 10 ml PGY medium containing peptone 5g/L, yeast extract using a BLX-E254 biolink (Vilber-Lourmat, France) of 4 2.5 g/L and, glucose 0.5g/L at 30 to 50°C. with aeration and mJ/cm2 each, carried out at an interval of 4 hours. After agitation. After 3 days the antibiotic production of the tested incubation at 30 to 50° C. for 3 to 4 days, the viable colonies strains was controlled and quantified by diffusion test agar of interest were visible. against several reference strains cultivated on LB medium. 0158 LB: Bacto-tryptone 10 g/L, Yeast extract 5 g/L, 0151 Examples of colonies identified are listed in Table 2 Sodium chloride 10 g/L below. 0159 PGY: peptone 5 g/L, yeast extract 2.5g/L, glucose Example 4 0.5g/L, (0160 Reference strains: Isolation of UV-Resistant Bacteria from Stones, 0.161 Staphylococcus aureus CIP 76.25 Moss, Lichen, Mud, Sediment, Biofilm, Soil and (0162 Escherichia coli CIP 76.24; Animal Dejection (0163 Candida tropicalis CIP 1275. The results are depicted in Table 3 below. 0152 Samples of moss, lichen, mud, soil and animal dejection were placed in suspension in sterile water (V/V) TABLE 3 then vortexed. The samples were then sonicated for approxi Tested Staphylococcus Escherichia coli Candida tropicalis mately 60 seconds. Bacteria attres CIP 76.25 CIP 76.24 CIP 1275 0153. Following sonication, between 150 ul and 2 ml of the Suspensions are spread on a solid PGY-agar enriched Strain 1 O -- -- Strain 2 -- -- O culture medium sterilized by autoclaving (20 minutes at 120° Strain 3 O O O C.) containing glucose (Sigma-Aldrich, France) 1 g/l, pep Strain 4 -- O O tone (Fluka, France) 10 g/l, and yeast extract (Fluka, France) Strain 5 -- O O 5 g/l. The seeded culture media then undergo 3 UV treatments Strain 6 O O -- using a BLX-E254 biolink (Vilber-Lourmat, France) of 4 Deinococcus O -- O mJ/cm2 each, carried out at an interval of 4 hours. After 0; no visible antibiosis; incubation at 30 to 50° C. for 3 to 4 days, the viable colonies +: visible antibiosis of interest are visible. US 2014/0017724 A1 Jan. 16, 2014

0164. The results show that several strains exhibiting anti (0173 They were cultivated on LB medium at 37° C. for 18 biotic activity have been isolated. These strains produce anti hours. Then, 10 mL soft agar (Luria Bertani medium with biotic activity which is active against Staphylococcus aureus, agar at 7 g/L) were inoculated at 2% (v/v) with preculture of Escherichia coli and/or Candida tropicalis. The producing target strains in a petri dish. strains are resistant to UV and can be grown at elevated temperature, thereby illustrating the efficacy of the method of Evaluation of Antibiotic Production this invention. 0.174. After incubation at 30° C. or 45° C. for 48 hours, 96 Example 7 hours, 168 hours and 336 hours, 1 mL-test culture was cen trifuged at 13000rpm for 3 minutes. The supernatant was then Antibiotic Production Screening Assay filtered on 0.22um-filter. 10 uL offiltered culture supernatant was inoculated on Soft agar containing target strains. After 7.1. Material and Method incubation at 37° C. for 18 hours, lysis plaques could be observed. Culture Media (0165 Different media were prepared to testantibiotic pro 7.2. Results duction. 0166 100 mL of solution A is composed by MnCl, 4H2O 0.175 Table 4 presents example of different obtained anti 0.1 g, ZnSO 7H2O 0.1 g and FeC1 0.1 g. biotic profiles. Kineococcus strain M10-5H produced antibi 0167 Medium 1 is composed by bacto peptone from otics against Gram positive test strains and E. coli CIP 76.24. casein 5g/L, bacto yeast extract 3 g/L and adjusted at pH 7.1. Deinococcus strain MCs,F produced antibiotics against S. 0168 Medium 2 is composed by cellobiose 10 g/L, aureus CIP 76.25. Microbacterium strain MA3-7G produced KHPO 1 g/L, MgSO 7HO 1 g/L, NaCl1 g/L, (NH), SO antibiotics against C. tropicalis CIP 1275 and Williamsia 2 g/L, CaCO 2 g/L and adjusted at pH 7.2. Solution A is strain MA3-6B produced antibiotics against Gram positive added at 1 ml/L. test strains and Paeruginosa CIP 76.110. TABLE 4

Results of antibiotic production tests

Staphylococcus Streptococcus Pseudomonas Escherichia Candida attreus CIP pneumonia aeruginosa coli CIP tropicalis 76.25 CIP 104.485 CIP 76.11.O 76.24 CIP 1275

Kineococcits ------M1O-SH Deinococcus -- MC5-7F Microbacterium - -- MA3-7G Williamsia ------MA3-6B

“+” means that an antibiosis has been observed. '-' means that no antibiosis has been observed.

0169 Medium 3 is composed by oatmeal 20 g/L and solu- Example 8 tion A at 1 ml/L. Production of Antioxidants by Deinococcus Culture Preparation of Test Strains 0176 The present example discloses the production of (0170 Strains were inoculated in 5 mL of PGY liquid antioxidants from Deinococcus. The example shows Deino media. Precultures were then incubated at 30° C. or 45° C. for coccus bacteria produce Substantial amounts of carotenoids, 48 hours under shaking at 150 rpm. which can be isolated and used as an antioxidant. Alterna 0171 In 15 mL-glass tube, 100 uL of preculture were tively, the whole bacteria, or extracts thereof, can be used as inoculated in 5 mL of media 1 to 3. Cultures were then antioxidant agent. incubated at 30°C. or 45° C. for 48 hours, 96 hours, 168 hours and 336 hours under shaking at 150 rpm. 8.1. Materials & Methods Culture Preparation of Target Strains 0177 Cultures of the Deinococcus strains were performed at 30°C., 150 rpm, in CMG medium containing glucose 1%. 0172 Target strains were Staphylococcus aureus CIP76. After, 48 hours, the culture is centrifuged to obtain a pellet, 25, Escherichia coli CIP 76.24, Pseudomonas aeruginosa the Supernatant being discarded. The extraction of caro CIP 76.110, Streptococcus pneumonia CIP 104485 and Can tenoids is performed with 3 washes of 1 ml methanol. Then, dida tropicalis DSM 1346. the quantity of carotenoids in the extraction fractions is deter US 2014/0017724 A1 Jan. 16, 2014 10 mined after reading OD at 480 nm with the following extinc 0183 These results show that all strains have an inhibitory tion coefficient of carotenoids (137000 M''cm). effect on the growth of eukaryotic cells HT29 and/or MRCS. Several culture fractions of Deinococcus strains M36-7D 8.2. Results (fractions n°9, 5 and 2) abolished totally the growth of both HT29 and MRCS eukaryotic cells when diluted up to 16-fold. (0178. The results are presented in the table below: In addition, Fractions n° 2 and 12 induce 100% of growth inhibition of MRCS cells when diluted to 4- and 8-fold, respectively. mg carotenoids. 0.184 Interestingly, culture fraction n° 9 of MA12-4C Strains g dry weight strain showed very good toxicity effect on HT29 cells even at Deinococci is radiopugnans 1.72 high dilution rate. These results therefore demonstrate that DSM 12027 Deinococcus produce efficient cytotoxic agents, which can be DRH11 Deinococcus M3-2D 1.01 isolated and used as active pharmaceutical ingredients. Deinococcus M1-8D O.47 Deinococciis M2-8C 0.44 Example 10

0179 These results show that Deinococcus strains pro Deinococcus Cultivation in Bioreactor for Industrial duce naturally high amounts of carotenoids that are known to Drug Production act as anti-oxidant. 0185. Deinococcus geothermalis is cultivated on R2YE Example 9 medium without Phosphate addition in 1 L-bioreactor (Bio stat Q+Sartorius). The temperature and pH is maintained at Production of Cytotoxic agents by Deinococcus or 45° C., and 6 respectively. The aeration is kept at 0.1 VVmand related bacteria pO2 at 20% with a cascade regulation on stirrer between 400 and 1000 rpm. The following composition of culture medium 0180. The present example discloses the production of R2YE: K-50, 1.23 mM: MgC1.6HO 50 mM. Glucose 55.5 cytotoxic agents from Deinococcus. The example shows mM (10 g/L); CaCl2.H2O 20 mM; ZnCl, 0.08 mg/L. FeCls. Deinococcus bacteria produce Substantial amounts of cyto 6HO 0.4 mg/L. CuCl2HO 0.02 mg/L: MnC14HO 0.02 toxic agents, which can be isolated and used as active phar mg/L. NaBO7.10H2O 0.02 mg/L. (NH4)Mo.O.4H2O maceutical ingredients. 0.02 mg/L, Yeast Extract 5 g/L. The drug can be isolated or purified using conventional techniques. 9.1. Material & Methods: 0181 Deinococcus strains M36-7D, MA3-11A, M45-6E REFERENCES and MA 12-4C were grown in PGY medium until stationary phase. Different samples have been taken throughout the 0186 Berdy J Bioactive microbial metabolites. J Antibiot growth and diluted at different concentrations (dilution fac (Tokyo). 2005 January: 58(1):1-26, Erratum in: J Antibiot tors: 1/4, 1/8, 1/16, 1/32, 1/64) and then tested for their tox (Tokyo). 2005 April; 58(4):C-1. icity against cancerous human cell lines HT29 and MRCS by 0187 Buchanan R and Gibbons N Bergey’s manual of using the in vitro toxicology assay kit, MTT based from determinative bacteriology (1974) 8th ed., DIFCO manual Sigma-Aldrich. The experiments have been performed Dehydrated Culture Media and Reagents for Microbiology according to the Sigma Aldrich toxicology assay protocol. (1995) Eds DIFCO, 11th ed. 0188 Ginolhac A. Jarrin C, Gillet B, Robe P. Pujic P. 9.2. Results Tuphile K, Bertrand H, Vogel T M. Perriere G. Simonet P. 0182. The results are presented in the following table. Nalin R Phylogenetic analysis of polyketide synthase I TABLE Percentage of inhibition of eukaryotic cells growth HT29 cells MRCS cells No Dilution factor

Strains fraction /4 /8 /16 1/32 /64 / /8 1/16 1/32 /64 Deinococcus M36-7D 9 100 nd ind O 1 ind 6 O O O geothermalis M36-7D 5 ind nd 100 35 15 O O O O O M36-7D 2 52 O 88 31 57 100 100 70 36 35 Microbacterium sp. MA3-11A 2 11 69 74 no ind 83 99 38 ind ind MA3-11A 12 19 S8 32 ind nd 100 0 0 ind ind Promicromonospora M45-6E 2 97 94 88 46 53 93 96 94 11 O M45-6E 3 91 93 87 66 S9 79 88 60 25 2 M45-6E 14 ind nd ind 4 6 100 O O O O Chronobacterium MA12-4C 3 91 74 79 83 89 O 1 O 24 27 24 haemolyticum MA12-4C 9 71. 89 92 96 94 1 3 85 79 8O MA12-4C 19 94 88 95 92 89 85 92 81 79 84 MA12-4C 22 87 94 92 97 86 96 9s 94 94 94 US 2014/0017724 A1 Jan. 16, 2014 11

domains from Soil metagenomic libraries allows selection chemistry—exodus or revival? Angew Chem Int Ed Engl. of promising clones. Appl Environ Microbiol. 2004 Sep 2006 Aug. 4:45(31):5072-129. tember: 70(9):5522-7. 0201 Yang B, Xue Q H, Chen ZQ, Zhou YQ, Zhang XL, 0189 Hamaki T, Suzuki M., Fudou R, Jojima Y. Kajiura T. Xu Y J, Guo ZY Effects of microwave irradiation on Tabuchi A, Sen K, Shibai HIsolation of novel bacteria and isolation of soil actinomycetes, Ying Yong Sheng Tai Xue actinomycetes using soil-extract agar medium. J Biosci Bao. 2008 May: 19(5):1091-8. Bioeng. 2005 May: 99(5):485-92. We claim: (0190. Hirsch P. GallikowskiCA, Siebert J. Peissl K, Krop 1. A method for producing a drug, comprising (i) culturing, penstedt R. Schumann P. Stackebrandt E. Anderson R in a suitable medium, a bacterium which naturally produces Deinococcus frigens sp. nov. Deinococcus saxicola sp. said drug oran intermediate thereof, (ii) collecting or purify nov., and Deinococcus marmoris sp. nov., low temperature ing the drug or intermediate from the culture, and (iii) when and draught-tolerating, UV-resistant bacteria from conti an intermediate is produced in step (i), converting said inter nental Antarctica. Syst Appl Microbiol. 2004 November; mediate into said drug, wherein said bacterium is a Deino 27(6):636-45. coccus or a related bacterium. (0191 Janssen P. Yates P. Grinton B, Taylor P. and Sait M 2. An improved method for the production of a drug from a Improved Culturability of Soil cultured microbial cell, the improvement consisting in the (0192 Bacteria and Isolation in Pure Culture of Novel use, as said microbial cell, of a Deinococcus or related bac Members of the Divisions Acidobacteria, Actinobacteria, terium. Proteobacteria, and Verrucomicrobia Applied and Environ 3. The method of claim 1, wherein the bacterium is a mental Microbiology, May 2002, p. 2391-2396, Vol. 68, No. Deinococcus bacterium. 5. (0193 Kämpfer P. Lodders N, Huber B, Falsen E. Busse H 4. The method of claim 1, wherein the bacterium is a J Deinococcus aquatilis sp. nov., isolated from water. IntJ wild-type Deinococcus bacterium. Syst Evol Microbiol. 2008 December; 58(Pt 12):2803-6. 5. The method of claim 1, wherein the bacterium is a Lorian V(ed.), Antibiotics in laboratory medicine. Will recombinant Deinococcus bacterium. iams & Wilkins, Baltimore, Md. 1996. 6. The method of claim 1, wherein the bacterium is cultured 0194 Makarova KS, Aravind L. WolfY I, Tatusov RL, in a defined medium. Minton K W. Koonin E. V. Daly M J Genome of the 7. The method of claim 1, wherein the bacterium is cultured extremely radiation-resistant bacterium Deinococcus in a complex medium. radiodurans viewed from the perspective of comparative 8. The method of claim 1, wherein the bacterium is cultured genomics MicrobiolMol Biol Rev. 2001 March; 65(1):44 at a pH comprised between 4 and 8. 79. 9. The method of claim 1, wherein the bacterium is cultured (0195 Marsden A. F. Wilkinson B, Cortes J. Dunster NJ, in a fermentation device or as a continuous culture. Staunton J. Leadlay P F Engineering broader specificity 10. The method of claim 1, wherein the drug is selected into an antibiotic-producing polyketide synthase. Science. from an antibiotic, a bacteriostatic agent, an anti-parasitic 1998 Jan. 9; 279(5348): 199-202. agent, an anti-fungal agent, an anti-Viral agent, an anti-me (0196. Rainey F. Ray K, Ferreira M, Gatz B, Nobre M, tabolite agent, a chemotherapeutic agent, a cytokine, a cell Bagaley D. Rash B, Park M. Earl A. Shank N. Small A, growth factor, a hormone, an antioxidant, an anti-inflamma Henk M. Battista J., Kämpfer P, and da CostaM Extensive tory agent, an enzyme inhibitor, a polysaccharide or an Diversity of Ionizing-Radiation-Resistant Bacteria Recov adjuvant. ered from Sonoran Desert Soil and Description of Nine 11. The method of claim 1, wherein the drug is an antibi New Species of the Genus Deinococcus Obtained from a otic. Single Soil Sample Appl Environ Microbiol. 2005 Septem 12. The method of claim 10, wherein the antioxidant is a ber 71(9): 5225-5235. carotenoid compound. (0197) Reichenbach H Myxobacteria, producers of novel 13. The method of claim 1, wherein the drug is chemically bioactive substances. J Ind Microbiol Biotechnol. 2001 modified after collection or purification. September; 27(3):149-56. 14. A method for producing a drug by transformation of a (0198 Schoenborn L. Yates P. Grinton B, Hugenholtz P. precursor thereof, said method comprising exposing the pre and Janssen P Liquid Serial Dilution Is Inferior to Solid cursor to a catalyst and collecting the drug resulting from the Media for Isolation of Cultures Representative of the Phy lum-Level Diversity of Soil Bacteria Applied and Environ transformation, wherein the catalyst is a Deinococcus or a mental Microbiology, July 2004, p. 4363-4366, Vol. 70, related bacterium, or an extract thereof. No. 7. 15. A pharmaceutical composition comprising a Deinococ (0199 Singh SB, Pelaez FBiodiversity, chemical diversity cus bacterium as defined in claim 1. and drug discovery. Prog Drug Res. 2008: 65:141, 143-74. 16. A composition comprising a Deinococcus bacterium as (0200 von Nussbaum F, Brands M, Hinzen B, Weigand S, an intestinal flora modifier. Habich D Antibacterial natural products in medicinal k k k k k