JP6435462B2 - Microbial culture method - Google Patents
Microbial culture method Download PDFInfo
- Publication number
- JP6435462B2 JP6435462B2 JP2014069424A JP2014069424A JP6435462B2 JP 6435462 B2 JP6435462 B2 JP 6435462B2 JP 2014069424 A JP2014069424 A JP 2014069424A JP 2014069424 A JP2014069424 A JP 2014069424A JP 6435462 B2 JP6435462 B2 JP 6435462B2
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- Prior art keywords
- microorganism
- culture
- microorganisms
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- antibiotic
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明は、微生物の培養方法に関し、より詳しくは、非優占的な微生物を分離して培養するための方法に関する。また、本発明は、新規な微生物をスクリーニングするための方法にも関する。 The present invention relates to a method for culturing a microorganism, and more particularly to a method for separating and culturing a non-dominant microorganism. The invention also relates to methods for screening novel microorganisms.
地球上に存在する微生物は、ＤＮＡレベルの解析の成果により１００万種以上存在すると言われている。しかしながら、同定されているのは約９千種と、その１割にも満たない。また、腸内に定着している微生物（腸内常在菌）においても７０％以上が未同定、未培養のままである。 Microorganisms present on the earth are said to be present in excess of one million by the results of DNA level analysis. However, about 9,000 species have been identified, less than 10% of them. In addition, 70% or more of the microbes established in the intestine (enteric indigenous bacteria) are unidentified and uncultured.
かかる現状において、培養操作を伴わないで環境中の微生物の核酸抽出から分子生物学的に解析する手法（メタゲノム的アプローチ）に着目されている。しかしながら、微生物の本質を理解し、また商業的価値を見出すためには、微生物を種毎に分離培養して解析する手法を用いた様々な検討が必須である。従って、微生物を産業上利用していく上では、メタゲノム的アプローチのみでは限界がある。さらに昨今の環境破壊等からこれらの微生物を保護する上でも、分離培養技術は普遍的に必要不可欠である。 Under the present circumstances, attention is focused on a method (metagenomic approach) of molecular biological analysis from nucleic acid extraction of microorganisms in the environment without culture operation. However, in order to understand the nature of microorganisms and to find commercial value, various studies using techniques for separating and culturing microorganisms for each species are essential. Therefore, there are limits to using metagenomic approaches alone in industrial use of microorganisms. Furthermore, in order to protect these microorganisms from recent environmental destruction etc., separation culture technology is universally necessary.
また、前述の通り、ヒトの腸内常在菌においては、未だ７割以上は未培養・未同定であるので、培養技術を改良することで未知の微生物の分離・同定を可能とすれば、新規な有用微生物（プロバイオティクス）が発見される可能性は非常に高い。そして、これら微生物又はこれらの代謝産物を活用することにより。病気の予防法や治療法の開発等も可能となる。 Furthermore, as described above, in human intestinal indigenous bacteria, 70% or more are still uncultured / unidentified, so if improvement and improvement of culture technology enable separation / identification of unknown microorganisms, It is very likely that new useful microorganisms (probiotics) will be discovered. And by utilizing these microorganisms or these metabolites. It will also be possible to develop methods for preventing and treating diseases.
しかしながら、腸内常在菌等の微生物の分離培養において、比較的菌数の少ない非優占的な微生物は一般的に、菌数の多い優占的な微生物の増殖の影響により、その増殖が抑制される傾向にある（非特許文献１）。また、非優占的な微生物は、培養や単分離が困難なゆえに、その多くは未だ同定されていないことが予想され、さらには、非優占的な微生物の中には、ヒトにとって有用な微生物も含まれている可能性は、十二分に考えられる。 However, in the separation and culture of microorganisms such as intestinal indigenous bacteria, non-dominant microorganisms having a relatively small number of bacteria generally grow as a result of the growth of dominant microorganisms having a large number of bacteria. There is a tendency to be suppressed (Non-Patent Document 1). In addition, it is expected that many non-dominant microorganisms have not yet been identified because they are difficult to culture or isolate, and some non-dominant microorganisms are useful for humans. The possibility that microorganisms are also included is considered more than enough.
微生物の分離培養を目的として、特定の抗生物質を添加した寒天培地を用いた塗抹培養が一般的に行われているが、非優占的な微生物にも添加した抗生物質が作用してしまい、選択した菌属の中でも菌数の多い種（優占種）のみしか分離できていないと考えられている。 Although smear culture using an agar medium to which a specific antibiotic is added is generally carried out for the purpose of separation culture of microorganisms, the antibiotic which has been added also acts on non-dominant microorganisms, It is thought that only species having a large number of fungi (dominant species) can be isolated among the selected genus of bacteria.
本発明は、前記従来技術の有する課題に鑑みてなされたものであり、非優占的な微生物の分離培養を可能とする方法、ひいては新規な微生物をスクリーニングするために方法を提供することを目的とする。 The present invention has been made in view of the problems of the prior art, and it is an object of the present invention to provide a method capable of separating and culturing non-dominant microorganisms, and thus a method for screening novel microorganisms. I assume.
本発明者らは、非優占的な微生物の増殖の阻害要因となる優占的な微生物の増殖を限定的に抑制し、非優占的な微生物の増殖を促すことを着想した。具体的には、図１に示すように、微生物群中の非優占的な微生物（図中「菌Ｃ」及び「菌Ｄ」）を培養する方法として、増殖速度の速い優占的な微生物（図中「菌Ａ」及び「菌Ｂ」）の対数増殖期に、当該微生物群を、細胞の増殖に必須の過程をブロックする抗生物質を複数種組み合わせて処理しながら培養することを想到した。そして、ヒトの新鮮糞便の希釈液（腸内常在菌群）を、当該腸内常在菌群が抗生物質非存在下にて対数増殖する期間において、前記腸内常在菌群を、細胞の増殖に必須の過程をブロックする抗生物質を複数種組み合わせて処理しながら、液体培養することにより、非優占的な微生物を分離培養できることを明らかにした。さらに、得られた非優占的な微生物の中から、今まで同定されていなかった新規の菌株を多数、同定することもでき、本発明を完成するに至った。 The present inventors have conceived of limiting the growth of dominant microorganisms, which is a factor that inhibits the growth of non-dominant microorganisms, and promoting the growth of non-dominant microorganisms. Specifically, as shown in FIG. 1, a dominant microorganism having a high growth rate is used as a method of culturing non-dominant microorganisms (“fungus C” and “fungus D” in the figure) in the microorganism group. In the logarithmic phase of growth ("Funga A" and "Fung B" in the figure), it was conceived that the microorganism group was cultured while being treated with a combination of two or more antibiotics that block a process essential for cell growth. . Then, a dilution solution of human fresh feces (enteric indigenous bacteria group) is used as the intestinal resident bacteria group in a period during which the intestinal resident bacteria group grows logarithmically in the absence of an antibiotic. It has been revealed that non-dominant microorganisms can be isolated and cultured by liquid culture while treating a combination of antibiotics that block processes essential for the growth of Furthermore, among the obtained non-dominant microorganisms, a large number of novel strains that have not been identified so far can be identified, and the present invention has been completed.
すなわち、本発明は以下を提供するものである。
＜１＞ 微生物群から特定の微生物を分離して培養するための方法であって、
前記微生物群が抗生物質非存在下にて対数増殖する期間の一部又は全部において、前記微生物群を抗生物質存在下にて液体培養する工程と、
前記工程にて液体培養した微生物群を、前記抗生物質非存在下にて分離培養する工程とを含み、
かつ、前記抗生物質が、核酸合成阻害剤、タンパク質合成阻害剤、細胞壁合成阻害剤及び葉酸合成阻害剤からなる群から選択される少なくとも２種の抗生物質の組み合わせである方法。
＜２＞ 前記抗生物質が、核酸合成阻害剤、タンパク質合成阻害剤、細胞壁合成阻害剤及び葉酸合成阻害剤の組み合わせである、＜１＞に記載の方法。
＜３＞ 前記抗生物質が、ノルフロキサシン、ホスホマイシン、テトラサイクリン及びトリメトプリムの組み合わせである、＜１＞に記載の方法。
＜４＞ 前記分離培養が塗抹培養である、＜１＞〜＜３＞のうちのいずれか１に記載の方法。
＜５＞ 前記微生物群が腸内常在菌群である、＜１＞〜＜４＞のうちのいずれか１に記載の方法。
＜６＞ 特定の微生物を生産するための方法であって、
＜１＞〜＜５＞のうちのいずれか１に記載の方法によって、微生物群から前記特定の微生物を分離して培養する工程と、
前記工程にて分離培養した前記特定の微生物を回収する工程と
を含む方法。
＜７＞ 微生物群から新規な微生物をスクリーニングするための方法であって、
＜１＞〜＜５＞のうちのいずれか１に記載の方法によって、微生物群から前記特定の微生物を分離して培養する工程と、
前記工程にて分離培養した前記特定の微生物の特性を分析する工程と、
前記工程にて得られた特性と公知の微生物の特性とを比較し、一致しない場合には、前記特定の微生物は新規な微生物であると判定する工程と
を含む方法。
That is, the present invention provides the following.
<1> A method for separating and culturing a specific microorganism from a microorganism group,
Liquid culturing the microorganism group in the presence of an antibiotic during part or all of a period during which the microorganism group grows logarithmically in the absence of the antibiotic;
Separating and culturing the microorganism group liquid-cultured in the step, in the absence of the antibiotic,
And, the method wherein the antibiotic is a combination of at least two antibiotics selected from the group consisting of a nucleic acid synthesis inhibitor, a protein synthesis inhibitor, a cell wall synthesis inhibitor and a folate synthesis inhibitor.
<2> The method according to <1>, wherein the antibiotic is a combination of a nucleic acid synthesis inhibitor, a protein synthesis inhibitor, a cell wall synthesis inhibitor and a folate synthesis inhibitor.
<3> The method according to <1>, wherein the antibiotic is a combination of norfloxacin, fosfomycin, tetracycline and trimethoprim.
<4> The method according to any one of <1> to <3>, wherein the separation culture is a smear culture.
<5> The method according to any one of <1> to <4>, wherein the microorganism group is a gut indigenous bacteria group.
<6> A method for producing a specific microorganism,
Separating and culturing the specific microorganism from the microorganism group by the method according to any one of <1> to <5>;
Recovering the specific microorganism separated and cultured in the step.
<7> A method for screening a novel microorganism from a microorganism group,
Separating and culturing the specific microorganism from the microorganism group by the method according to any one of <1> to <5>;
Analyzing the characteristics of the specific microorganism separated and cultured in the step;
And D. comparing the property obtained in the step with the property of a known microorganism, and if not identical, determining that the specific microorganism is a novel microorganism.
本発明によれば、非優占的な微生物を分離培養することが可能となり、ひいては、非優占的な微生物の中から、新規な微生物を同定することも可能となる。 According to the present invention, it becomes possible to separate and culture non-dominant microorganisms, and it is also possible to identify new microorganisms among non-dominant microorganisms.
＜微生物の培養方法＞
本発明の微生物の培養方法は、微生物群から特定の微生物を分離して培養するための方法であって、
前記微生物群が抗生物質非存在下にて対数増殖する期間の一部又は全部において、前記微生物群を抗生物質存在下にて液体培養する工程と、
前記工程にて液体培養した微生物群を、前記抗生物質非存在下にて分離培養する工程とを含み、
かつ、前記抗生物質が、核酸合成阻害剤、タンパク質合成阻害剤、細胞壁合成阻害剤及び葉酸合成阻害剤からなる群から選択される少なくとも２種の抗生物質の組み合わせである方法である。
<Method of culturing microorganisms>
The culture method of the microorganism of the present invention is a method for separating and culturing a specific microorganism from a microorganism group,
Liquid culturing the microorganism group in the presence of an antibiotic during part or all of a period during which the microorganism group grows logarithmically in the absence of the antibiotic;
Separating and culturing the microorganism group liquid-cultured in the step, in the absence of the antibiotic,
And, the method wherein the antibiotic is a combination of at least two antibiotics selected from the group consisting of a nucleic acid synthesis inhibitor, a protein synthesis inhibitor, a cell wall synthesis inhibitor and a folate synthesis inhibitor.
本発明における「微生物」は、肉眼では観察が困難な微小な生物の意として用いられ、その多くは１ｍｍ以下の体長である。生物種としては特に制限されることなく、例えば、微細藻類、原生動物、真菌、粘菌等の真核生物、及び真正細菌、古細菌等の原核生物が挙げられる。 The "microbe" in the present invention is used as the meaning of a minute organism which is difficult to observe with the naked eye, and most of them are 1 mm or less in length. The biological species is not particularly limited, and examples thereof include microalgae, protozoa, eukaryotes such as fungi and slime molds, and prokaryotes such as eubacteria and archaebacteria.
本発明における「特定の微生物」とは、特に、後述の抗生物質非存在下における液体培養において、菌数の多い微生物（以下、「優占的な微生物」とも称する）の増殖の影響により、その増殖が抑制される傾向にある、比較的菌数の少ない微生物（以下、「非優占的な微生物」とも称する）のことを意味するまた、「特定の微生物」は、特定の一種の微生物であってもよく、特定の複数種の微生物であってもよい。 In the present invention, the “specific microorganism” is, in particular, the influence of the growth of a microorganism having a large number of bacteria (hereinafter also referred to as “dominant microorganism”) in liquid culture in the absence of antibiotics described later. It means a relatively small number of microorganisms (hereinafter also referred to as "non-dominant microorganisms") whose growth tends to be suppressed, and "specific microorganism" is a specific type of microorganism. It may be or may be a plurality of specific types of microorganisms.
本発明における「微生物群」とは、前記特定の微生物を含む、微生物の集団を意味し、例えば、宿主生物（ヒト等の哺乳動物、鳥類、ハ虫類、両生類、魚類、甲殻類、昆虫類、貝類、軟体動物、植物等）の体内若しくは体表に、又は排泄物、滲出物、分泌物若しくは体液に存在する微生物の集団（腸内常在微生物叢（腸内常在菌群）、口腔内微生物叢、皮膚微生物叢等）が挙げられる。さらには、海洋（海水）、河川・湖（淡水）、熱水、土壌、堆積物、大気圏、地殻内等の自然環境内に存在する微生物叢も、本発明における「微生物群」として挙げられる。また、本発明において、「微生物群」としては、その形態に特に制限はなく、微生物のみの集団であってもよく、該集団を含む試料（例えば、腸内常在菌群を含む糞便試料、土壌微生物叢を含む土壌試料）であってもよい。さらには、当該試料を水、生理食塩水又は緩衝液等にて希釈して得られる懸濁液であってもよく、また、該試料の培養物（該試料を培養して得られる培養液、培地等を含む）であってもよい。 The “microbe group” in the present invention means a group of microorganisms including the above-mentioned specific microorganism, for example, host organisms (eg mammals such as humans, birds, reptiles, amphibians, fish, crustaceans, insects) , Shellfish, molluscs, plants, etc.), or excrement, exudate, secretion, or population of microorganisms present in body fluid (enteric indigenous microflora (enteric indigenous bacteria group), oral cavity Internal microflora, skin microflora etc.). Furthermore, the microflora present in natural environments such as the ocean (seawater), rivers and lakes (fresh water), hot water, soil, sediments, atmosphere, crust and the like are also mentioned as “microbe group” in the present invention. Further, in the present invention, the “microbe group” is not particularly limited in its form, and may be a population of only microorganisms, and a sample containing the population (eg, a fecal sample including a gut resident population, It may be a soil sample containing a soil microflora. Furthermore, it may be a suspension obtained by diluting the sample with water, physiological saline, buffer or the like, and a culture of the sample (a culture solution obtained by culturing the sample, The medium may be included.
本発明において「抗生物質」は、微生物を殺すか、微生物の増殖又は機能を阻害する薬剤を意味し、微生物の産生物に由来する天然の薬剤のみならず、人工的に合成された薬剤も含む。さらに、本発明において「抗生物質」は、下記に示す４種の抗生物質：核酸合成阻害剤、タンパク質合成阻害剤、細胞壁合成阻害剤及び葉酸合成阻害剤からなる群から選択される少なくとも２種の抗生物質の組み合わせである必要があり、好ましくは、ＤＮＡ合成阻害剤、タンパク質合成阻害剤及び細胞壁合成阻害剤の組み合わせであり、より好ましくは、ＤＮＡ合成阻害剤、タンパク質合成阻害剤、細胞壁合成阻害剤及び葉酸合成阻害剤の組み合わせであり、特に好ましくは、ノルフロキサシン、テトラサイクリン、ホスホマイシン及びトリメトプリムの組み合わせである。 In the present invention, "antibiotic" means an agent that kills a microorganism or inhibits the growth or function of a microorganism, and includes not only natural agents derived from the products of microorganisms but also artificially synthesized agents . Furthermore, in the present invention, the “antibiotic” is at least two selected from the group consisting of four types of antibiotics shown below: nucleic acid synthesis inhibitors, protein synthesis inhibitors, cell wall synthesis inhibitors and folate synthesis inhibitors It needs to be a combination of antibiotics, preferably a combination of a DNA synthesis inhibitor, a protein synthesis inhibitor and a cell wall synthesis inhibitor, more preferably a DNA synthesis inhibitor, a protein synthesis inhibitor, a cell wall synthesis inhibitor And a combination of folate synthesis inhibitors, particularly preferably a combination of norfloxacin, tetracycline, fosfomycin and trimethoprim.
（１）核酸合成阻害剤
ＤＮＡジャイレース阻害作用等の核酸合成を阻害する作用を有する抗生物質であり、例えば、ノルフロキサシン、オフロキサシン、エノキサシン、塩酸シプロフロキサシン、塩酸ロメフロキサシン、レボフロキサシン、ガレノキサシン、フレロキサシン、シタフロキサシン、トスフロキサシントシル酸塩、スパルフロキサシン、ガチフロキサシン、モキシフロキサシン等のキノロン系抗生物質、リファンピシン等のリファンピシン系抗生物質が挙げられる。
(1) Nucleic acid synthesis inhibitor An antibiotic having an activity of inhibiting nucleic acid synthesis such as DNA gyrase inhibitory activity, such as norfloxacin, ofloxacin, enoxacin, ciprofloxacin hydrochloride, lomefloxacin hydrochloride, levofloxacin, galenoxacin, freloxacin, Examples thereof include quinolone antibiotics such as sitafloxacin, tosufloxacin tosilate, sparfloxacin, gatifloxacin, moxifloxacin and the like, and rifampicin antibiotics such as rifampicin.
（２）タンパク質合成阻害剤
タンパク質の合成を阻害する作用を有する抗生物質であり、例えば、テトラサイクリン、ドキシサイクリン、ミノサイクリン、オキシテトラサイクリン、デメクロサイクリン、クロルテトラサイクリン等のテトラサイクリン系抗生物質、ストレプトマイシン、カナマイシン、ゲンタマイシン、トブラマイシン、アミカシン、ジベカシン、アルべカシン等のアミノグリコシド系抗生物質、エリスロマイシン、クラリスロマイシン、ロキシスロマイシン、アジスロマイシン、ジョサマイシン、ロキタマイシン、キタサマイシン、アセチルスピラマイシン、テリスロマイシン、リンコマイシン、クリンダマイシン、
タクロリムス等のマクロライド・ケトライド系抗生物質、クロラムフェニコール等のクロラムフェニコール系抗生物質が挙げられる。
(2) Protein synthesis inhibitor An antibiotic having an activity of inhibiting protein synthesis, for example, tetracycline, such as tetracycline, doxycycline, minocycline, minocycline, oxytetracycline, demecrocycline, chlortetracycline, streptomycin, kanamycin, gentamycin Aminoglycoside antibiotics such as tobramycin, amikacin, dibekacin, arbekacin, erythromycin, clarithromycin, roxithromycin, azithromycin, josamycin, lokitamycin, kitasamycin, acetylspyramycin, telithromycin, lincomycin, clindamycin,
Macrolide ketolide antibiotics such as tacrolimus and chloramphenicol antibiotics such as chloramphenicol.
（３）細胞壁合成阻害剤
細胞壁の合成を阻害する作用を有する抗生物質であり、例えば、ホスホマイシン等のホスホマイシン系抗生物質、ペニシリン、ベンジルペニシリン、フェノキシメチルペニシリン、ベンジルペニシリンベンザチン、メチシリン
、オキサシリン、クロキサシリン、ジクロキサシリン、アンピシリン、アモキシシリン、バカンピシリン、タランピシリン、カルベニシリン、チカルシリン、メズロシリン、テモシリン、アパラシリン、ピペラシリン、スルタミシリン、アゾシリン、ピブメシリナム等のペニシリン系抗生物質、セファゾリン、セファロチン、セファピリン、セファレキシン、セファラジン、セファドロキシル、セフマンドール、セフロキシム、セフォニシド、セフォラニド、セファクロル、セフプロジル、セフポドキシム、ロラカルベフ、セフトリアキソン、セフォタキシム、セフチゾキシム、セフタジジム、セフォペラゾン、セフスロジン、セフチブテン、セフィキシム、セフェタメット、セフジトレン
ピボキシル、セフェピム、セフピロム、セフォキシチン、セフォテタン、セフメタゾール、セフブペラゾン、セフミノクス、ラタモキセフ、フロモキセフ等のセフェム・オキサセフェム系抗生物質、ファロペネム、イミペネム、メロペネム等のペネム・カルバペネム系抗生物質、アズトレオナム、カルモナム等のモノバクタム系抗生物質が挙げられる。
(3) Cell wall synthesis inhibitor An antibiotic having an action to inhibit the synthesis of cell wall, for example, fosfomycin antibiotics such as fosfomycin, penicillin, benzylpenicillin, phenoxymethylpenicillin, benzylpenicillin benzathine, methicillin, oxacillin, cloxacillin , Penicillin antibiotics such as dicloxacillin, ampicillin, amoxicillin, bacampicillin, carbenicillin, carbicillin, ticarcillin, mezlocillin, temocillin, apalacillin, apalacillin, piperacillin, sultamicillin, azocillin, azocillinum, pibumesilinam, cefazolin, cefarotin, cephapirin, cepharidine, cefam fifamium, sev , Cefonicide, cefranide, cefaclor, cefprozil, sefa Fuphodoxime, Loracarbef, Ceftriaxone, Cefotixim, Cefizoxim, Cefazidime, Cefoperazone, Cefsurosin, Ceftiobuten, Cefoxime, Cefetame, Cefetame Pivoxil, Cefpirome, Cefoxitin, Cefotetan, Cefotiphere Teutraheavihecavietahe Examples thereof include penem and carbapenem antibiotics such as oxacephem antibiotics, faropenem, imipenem and meropenem, and monobactam antibiotics such as aztreonam and carmonam.
（４）葉酸合成阻害剤
葉酸の合成（代謝）を阻害する作用を有する抗生物質であり、例えば、トリメトプリム等のトリメトプリム系抗生物質、プロントジル、スルファモノメトキシン、スルファジアジン、スルファジメトキシン、スルファセタミド、スルファドキシン、スルファニルアミド、スルフィソミジン、スルフィソキサゾール、スルファメトキサゾール、スルファジミジン、スルファメラジン、スルファキノキサリン等のスルホンアミド系抗生物質が挙げられる。
(4) Folate synthesis inhibitor An antibiotic having an action to inhibit the synthesis (metabolism) of folate, for example, trimethoprim antibiotics such as trimethoprim, prontodil, sulfamonomethoxine, sulfadiazine, sulfadimethoxine, sulfacetamide, sulfado And sulfonamide antibiotics such as xin, sulfanylamide, sulfisomidine, sulfisoxazole, sulfamethoxazole, sulfadimidine, sulfamelazine, sulfaquinoxaline and the like.
本発明の微生物の培養方法においては、先ず、前記微生物群が抗生物質非存在下にて対数増殖する期間の一部又は全部において、前記微生物群を抗生物質存在下にて液体培養する。 In the method for culturing a microorganism of the present invention, first, the microorganism group is liquid-cultured in the presence of an antibiotic during part or all of the period in which the microorganism group logarithmically grows in the absence of the antibiotic.
本発明において「液体培養」とは、培養液中にて微生物を培養することを意味する。この液体培養に用いられる培養液としては特に制限はなく、培養する微生物群又は特定の微生物に合わせて、適宜、様々な物質を添加することにより調製することができる。例えば、窒素源として、アンモニア、塩化アンモニウム、硫酸アンモニウム、酢酸アンモニウム、リン酸アンモニウム等の無機酸若しくは有機酸のアンモニウム塩、牛肉・魚肉等を原料とした肉エキス、ポテト等を原料とした植物エキス、パン酵母・ビール酵母等を原料とした酵母エキス、コーンスチープリカー、大豆粕及び大豆粕加水分解物、各種発酵菌体並びにその消化物、その他の含窒素化合物等を培養液に添加してもよい。また、カゼインペプトン、獣肉ペプトン、心筋ペプトン、ゼラチンペプトン、大豆ペプトン等に例示されるペプトン類が培養液に添加されていてもよい。さらに、炭素源として、グルコース、フラクトース、スクロース、これらを含有する糖蜜、デンプンあるいはデンプン加水分解物等の炭水化物、酢酸、プロピオン酸等の有機酸、エタノール、プロパノール等のアルコール類等も培養液に添加されていてもよく、無機塩として尿素、アンモニア、硫酸アンモニウム、塩化アンモニウム、リン酸アンモニウム、リン酸第一カリウム、リン酸第二カリウム、塩化カリウム、水酸化カリウム、過リン酸石灰、リン安、リン酸マグネシウム、塩酸マグネシウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガン、硫酸銅、炭酸カルシウム等のリン酸成分、カリウム成分、マグネシウム成分、その他、亜鉛、銅、マンガン、鉄イオン等も培養液に添加されていてもよい。その他、ビタミン、核酸関連物質等も培養液に添加されていてもよい。さらに、微生物の生息環境に近い環境を再現させるために、生息環境であった土壌や堆積物由来の成分、海水、淡水等を培養液に適宜添加してもよい。また、宿主生物内又は表面での生息環境を再現するために、宿主生物（ヒト等の哺乳動物、鳥類、ハ虫類、両生類、魚類、甲殻類、昆虫類、貝類、軟体動物、植物等）由来の排泄物、滲出物、分泌物、体液、組織抽出物等、又は、酵母若しくは植物の抽出物等を培養液に適宜添加してもよい。さらに、かかる培養液のｐＨは、培養する微生物群又は特定の微生物に合わせて、適宜調整することができる。また、本発明における液体培養の条件、例えば、温度、湿度、酸素濃度、二酸化炭素濃度の条件も、培養する微生物群又は特定の微生物に合わせて、適宜調整することができる。 In the present invention, “liquid culture” means culturing a microorganism in a culture solution. There is no restriction | limiting in particular as a culture solution used for this liquid culture, According to the microbe group to culture | cultivate or a specific microbe, it can prepare by adding various substances suitably. For example, as nitrogen sources, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, meat extracts made from beef and fish, etc., plant extracts made from potatoes, etc. Yeast extract, corn steep liquor, soybean meal and soybean meal hydrolysates, various fermented cells and their digested products, and other nitrogen-containing compounds may be added to the culture solution. . In addition, peptones exemplified by casein peptone, animal meat peptone, cardiac muscle peptone, gelatin peptone, soybean peptone and the like may be added to the culture solution. Furthermore, as a carbon source, glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, alcohols such as ethanol, propanol and the like are added to the culture solution Which may be inorganic salts such as urea, ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, potassium phosphate, potassium phosphate, potassium chloride, potassium hydroxide, calcium perphosphate, phosphorus phosphate, phosphorus Also cultured are phosphoric acid components such as magnesium acid, magnesium hydrochloride, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate etc., potassium component, magnesium component, others, zinc, copper, manganese, iron ions etc. It may be added to the solution. In addition, vitamins, nucleic acid related substances and the like may be added to the culture solution. Furthermore, in order to reproduce the environment close to the inhabiting environment of the microorganism, components derived from the inhabiting environment soil or sediment, seawater, fresh water, etc. may be added as appropriate to the culture solution. Also, in order to reproduce the habitat in or on the host organism, host organisms (mammals such as humans, birds, reptiles, amphibians, fish, crustaceans, insects, shellfish, molluscs, plants, etc.) Excrements, exudates, secretions, body fluids, tissue extracts, etc. derived from or yeast or plant extracts etc. may be added to the culture solution as appropriate. Furthermore, the pH of such a culture solution can be appropriately adjusted in accordance with the group of microorganisms to be cultured or a specific microorganism. In addition, the conditions for liquid culture in the present invention, for example, the conditions of temperature, humidity, oxygen concentration and carbon dioxide concentration, can be appropriately adjusted according to the group of microorganisms to be cultured or a specific microorganism.
本発明にかかる液体培養において、前記抗生物質を培養液に添加しておく期間としては、前記微生物群が抗生物質非存在下にて対数増殖する期間の一部又は全部である。 In the liquid culture according to the present invention, a period during which the antibiotic is added to the culture solution is a part or all of a period during which the microorganism group grows logarithmically in the absence of the antibiotic.
「抗生物質の非存在下」とは、前記抗生物質の組み合わせが存在していない条件のことをいい、抗生物質が実質的に存在していない条件を意味する。また、優占的な微生物を死滅させるため抗生物質を長時間に渡って処理した場合、目的の非優占的な微生物も死滅させる恐れがある。そのため、本発明においては優占的な微生物群が抗生物質非存在下にて対数増殖する期間を対象にして抗生物質を処理する。「微生物群が抗生物質非存在下にて対数増殖する期間」とは、微生物群を抗生物質の非存在下にて液体培養した際に、培養時間に対して指数関数的に増殖する期間のことを意味する。さらに、後述の実施例において示す通り、前記抗生物質による処理時期及びその時間の違いによって、分離培養できる特定の微生物の種類は変動するため、所望の微生物が分離培養できるよう、前記抗生物質を培養液に添加しておく期間は、前記対数増殖する期間内において適宜調整され得る。当該期間としては、好ましくは抗生物質を培養液に添加してから１時間以上１８時間以内であり、より好ましくは２時間以上、さらに好ましくは４時間以上である。目的の非優占的な微生物の生存への影響を考慮して、より好ましく１５時間以内であり、より好ましくは１２時間以内、９時間以内、７時間以内である。また、前記抗生物質の培養液への添加濃度としては、培養する微生物群又は特定の微生物に合わせて、適宜調整することができる。
"In the absence of antibiotics" refers to conditions in which the combination of antibiotics is not present, and means conditions in which antibiotics are substantially absent. Also, if antibiotics are treated over a long period of time to kill dominant microorganisms, there is a risk that the target non-dominant microorganisms will also be killed. Therefore, in the present invention, antibiotics are treated for a period in which dominant microorganisms are grown logarithmically in the absence of antibiotics. The "period of logarithmic growth in the absence of antibiotics" is the period of exponential growth with respect to the culture time when the microorganisms are cultured in liquid in the absence of antibiotics. Means Furthermore, as shown in the examples described below, since the type of specific microorganism that can be separated and cultured varies depending on the treatment time with the antibiotic and the difference in the time thereof, the antibiotic is cultured so that the desired microorganism can be separated and cultured. The period of addition to the solution can be appropriately adjusted within the period of logarithmic growth. The period is preferably 1 hour or more and 18 hours or less, more preferably 2 hours or more, and still more preferably 4 hours or more after adding the antibiotic to the culture solution. It is more preferably within 15 hours, more preferably within 12 hours, within 9 hours, within 7 hours, in consideration of the influence on the survival of the objective non-dominant microorganism. The concentration of the antibiotic added to the culture solution can be appropriately adjusted according to the group of microorganisms to be cultured or a specific microorganism.
例えば、腸内常在菌群を、ノルフロキサシン、テトラサイクリン、ホスホマイシン及びトリメトプリムの存在下、ＮＴ培地にて液体培養する際には、これら抗生物質の好適な添加濃度としては、ノルフロキサシンを０．１〜３．１３μｇ／ｍＬ（但し、耐性菌Ｂ．ｆｒａｇｉｌｉｓでは２０〜１００μｇ／ｍＬ）、テトラサイクリンを０．８〜６．２５μｇ／ｍＬ（但し、耐性菌は８〜４０μｇ／ｍＬ以上）、ホスホマイシンは０．１２〜６４μｇ／ｍＬ（但し、耐性を持つものは１００〜５００μｇ／ｍＬ以上）、トリメトプリムを２〜１０μｇ／ｍＬ以上が挙げられる。 For example, when liquid cultures of enteropathogenic bacteria are carried out on NT medium in the presence of norfloxacin, tetracycline, fosfomycin and trimethoprim, the preferred addition concentration of these antibiotics is 0.1 to 3 for norfloxacin. .13 μg / mL (but 20 to 100 μg / mL for resistant B. fragilis), 0.8 to 6.25 μg / mL for tetracycline (however, 8 to 40 μg / mL or more for resistant bacteria), 0.12 for fosfomycin -64 micrograms / mL (However, those with tolerance are 100-500 micrograms / mL or more), 2-10 micrograms / mL or more of trimethoprim are mentioned.
このように、前記抗生物質存在下にて、前記微生物群の液体培養を、前記対数増殖する期間の一部又は全部において行うことにより、前記微生物群における優占的な微生物の対数増殖を抑制することができる。故に、本発明においては、かかる液体培養を行うことにより、当該対数増殖に影響を受けずに、特定の微生物の増殖は促進されるため、前記抗生物質非存在下にて、特定の微生物を分離培養することが可能となる。 Thus, by performing liquid culture of the group of microorganisms in part or all of the period of logarithmic growth in the presence of the antibiotic, the logarithmic growth of dominant microorganisms in the group of microorganisms is suppressed. be able to. Therefore, in the present invention, by performing such liquid culture, the growth of a specific microorganism is promoted without being affected by the logarithmic growth, so that the specific microorganism is separated in the absence of the antibiotic. It becomes possible to culture.
ここで「抗生物質非存在下」は前述の通りであり、かかる条件は、前述の抗生物質存在下にて培養した微生物群と、前記抗生物質を含有する培養液とを分離することにより、調製することができる。かかる微生物群と培養液とを分離する方法としては特に制限はなく、例えば、遠心分離、フィルター処理（通常、孔径が０．２２μｍ以下であるフィルターによる処理）が挙げられる。 Here, “in the absence of an antibiotic” is as described above, and such conditions are prepared by separating the microorganism group cultured in the presence of the above-mentioned antibiotic and the culture solution containing the above-mentioned antibiotic. can do. There is no restriction | limiting in particular as a method to isolate | separate this microorganisms group and a culture solution, For example, centrifugation, a filter process (The process by the filter whose pore diameter is 0.22 micrometer or less normally) is mentioned.
また、本発明における「分離培養」としては、前述の液体培養した微生物群における他の微生物と、特定の微生物とを分離して培養できればよく、このような培養は、例えば、限外希釈、混釈培養、塗抹培養によって行うことができる。 Furthermore, as “separate culture” in the present invention, it is sufficient that the specific microorganism can be separated and cultured from the other microorganisms in the liquid cultured microorganism group described above, and such culture can be carried out, for example, by limiting dilution or mixing. It can be performed by sham culture and smear culture.
本発明にかかる「限外希釈」においては、前述の液体培養した微生物群を前記培養液にて何倍にも希釈して、前記抗生物質非存在下にて培養することにより、単一の特定の微生物に由来する培養液を得ることができる。 In the "limiting dilution" according to the present invention, a single identification is carried out by diluting the above-mentioned liquid cultured microorganisms in the culture solution by many times and culturing in the absence of the antibiotic. A culture fluid derived from the microorganism of
本発明にかかる「混釈培養」においては、前述の液体培養した微生物群又はその一部を、固化する前の固形培地に加えて混釈し、固化させた後、前記抗生物質非存在下にて培養することにより、単一の特定の微生物に由来するコロニーを得ることができる。 In the “pouring culture” according to the present invention, the aforementioned liquid cultured microorganism group or a part thereof is added to the solid medium before solidification, mixed, solidified, and then in the absence of the antibiotic. By culturing it, colonies derived from a single specific microorganism can be obtained.
本発明にかかる「塗抹培養」においては、前述の液体培養した微生物群を白金耳等にとり、固形培地の表面上に塗抹し、前記抗生物質非存在下にて培養することにより、単一の特定の微生物に由来するコロニーを得ることができる。 In the "smear culture" according to the present invention, the aforementioned liquid cultured microorganism group is put on a platinum ear, etc., smeared on the surface of a solid medium, and cultured in the absence of the aforementioned antibiotic to identify a single one. It is possible to obtain colonies derived from the following microorganisms.
これら培養法においては、所望する菌株の単離の容易性の観点から、塗抹培養が好ましい。 In these culture methods, smear culture is preferable from the viewpoint of the ease of isolation of the desired strain.
また、混釈培養や塗抹培養に用いられる固形培地としては特に制限はなく、前記液体培養に用いられる培養液同様に、培養する微生物群又は特定の微生物に合わせて、前述の通り、窒素源、ペプトン類、炭素源、無機塩等の様々な物質を、固化させるために必要な寒天等と添加することにより調製することができる。かかる固形培地における寒天等の濃度は、培養する微生物群又は特定の微生物に合わせて、適宜調整することができるが、通常、０．３〜２．０％である。また、かかる固形培地のｐＨは、培養する微生物群又は特定の微生物に合わせて、適宜調整することができる。さらに、本発明にかかる混釈培養や塗抹培養の条件、例えば、温度、湿度、酸素濃度、二酸化炭素濃度の条件も、培養する微生物群又は特定の微生物に合わせて、適宜調整することができる。 Further, the solid medium used for pour culture and smear culture is not particularly limited, and as with the culture solution used for the liquid culture, according to the group of microorganisms to be cultured or a specific microorganism, as described above, a nitrogen source, It can be prepared by adding various substances such as peptones, carbon sources, inorganic salts and the like with agar necessary for solidifying. The concentration of agar or the like in such a solid medium can be appropriately adjusted according to the group of microorganisms to be cultured or a specific microorganism, but is usually 0.3 to 2.0%. In addition, the pH of such a solid medium can be appropriately adjusted according to the group of microorganisms to be cultured or a specific microorganism. Furthermore, conditions for mix culture and smear culture according to the present invention, for example, conditions of temperature, humidity, oxygen concentration, carbon dioxide concentration can be appropriately adjusted according to the group of microorganisms to be cultured or a specific microorganism.
以上、本発明にかかる分離培養の好適な実施形態について説明したが、本発明にかかる分離培養は上記実施形態に限定されるものではない。例えば、特定の微生物が、共生関係にある他の微生物（以下「共生微生物」とも称す）との共生下においてのみ生存・増殖することが可能な微生物、又は共生微生物との共生下において増殖が促進される微生物である場合には、図２に示すような培養システムを用い、下記培養方法にて分離培養することが好ましい。 As mentioned above, although the suitable embodiment of the separation culture concerning the present invention was described, the separation culture concerning the present invention is not limited to the above-mentioned embodiment. For example, growth is promoted under the symbiosis with a microorganism in which a specific microorganism can survive or grow only in symbiosis with other microorganisms in a symbiotic relationship (hereinafter also referred to as "symbiotic microorganism") or In the case of the microorganism to be isolated, it is preferable to perform separate culture by the following culture method using a culture system as shown in FIG.
共生微生物を第二の寒天培地で培養する工程と、
第一の寒天培地と第二の寒天培地を区画する工程であって、両寒天培地で培養される各微生物を通過させないが微生物が産出する物質は通過させるフィルターで区画する工程と、
前述の液体培養した微生物群を第一の寒天培地で培養する工程とを含む、
特定の微生物を共生微生物と共培養するための方法。
Culturing the symbiotic microorganism in a second agar medium;
Partitioning the first agar medium and the second agar medium with a filter that does not allow passage of each microorganism cultured in both agar media, but allows passage of a substance produced by the microorganism;
Cultivating the aforementioned liquid cultured microorganism group in a first agar medium,
Methods for co-cultivating specific microorganisms with symbiotic microorganisms.
かかる「特定の微生物を共生微生物と共培養するための方法」（以下「共培養法」とも称する）において、「微生物が産出する物質」としては特に制限はなく、例えば、微生物を構成する物質、微生物の分泌産物、微生物による代謝産物が挙げられる。 In the “method for cocultivating a specific microorganism with a symbiotic microorganism” (hereinafter also referred to as “coculture method”), the “substance produced by the microorganism” is not particularly limited, and, for example, a substance that constitutes the microorganism, They include secretory products of microorganisms and metabolic products of microorganisms.
かかる共培養法において、「フィルター」は、培養している微生物は通過させずに、それらが産出する物質を通過させるために用いられる。そのため、フィルターの孔径は、０．２２μｍ以下であることが好ましい。また、かかるフィルターの材質は特に制限されないが、親水性の材質が好ましい。 In such co-culture methods, "filters" are used to pass the material produced by them without passing through the culturing microorganisms. Therefore, the pore diameter of the filter is preferably 0.22 μm or less. Further, the material of the filter is not particularly limited, but a hydrophilic material is preferable.
「第一の寒天培地」は、前述の液体培養した微生物群における他の微生物から分離して、特定の微生物にシングルコロニーを形成させるために用いられる。そのため、「第一の寒天培地」は、該シングルコロニーが形成できる程度の固形性を保ち、かつ前記物質を拡散できる程度の流動性を保つ濃度の寒天を有している培地であればよい。かかる寒天の濃度としては、０．３〜１．０％の範囲であることが好ましい。 The "first agar medium" is used to separate specific microorganisms from other microorganisms in the liquid cultured microorganism group described above to form single colonies. Therefore, the "first agar medium" may be a medium having agar at a concentration that maintains solidity to such an extent that the single colony can be formed and maintains fluidity to such an extent that the substance can be diffused. The concentration of such agar is preferably in the range of 0.3 to 1.0%.
また、「第二の寒天培地」は、特定の微生物の生存・増殖に必要な因子を供給する為の培地であり、少なくとも共生微生物を培養して特定の微生物の生息環境を擬似的に再現することを目的に用いられる。そのため、「第二の寒天培地」は、共生微生物が産出する物質を拡散できる程度の流動性を保つ濃度の寒天であり、かつ培養システムを構築する際に構造的に安定する程度の固形性を保つ濃度の寒天を有している培地であればよい。かかる寒天の濃度としては、０．３〜１．０％の範囲であることが好ましい。 The "second agar medium" is a medium for supplying factors necessary for the survival and growth of a specific microorganism, and at least symbiotic microorganisms are cultured to simulate the habitat of the specific microorganism. It is used for the purpose. Therefore, the "second agar medium" is agar of a concentration that maintains the fluidity to such an extent that the substance produced by the symbiotic microorganism can be diffused, and the solidity to a degree that provides structural stability when constructing the culture system. Any medium having a concentration of agar to be maintained may be used. The concentration of such agar is preferably in the range of 0.3 to 1.0%.
また、第二の寒天培地において共生微生物を培養する際には、当該微生物以外に、特定の微生物と共生関係にない微生物を混入して培養してもよい。すなわち、第二の寒天培地においては、共生微生物を含む微生物群、前述の液体培養した微生物群等を培養してもよい。 When the symbiotic microorganism is cultured in the second agar medium, a microorganism which is not in a symbiotic relationship with a specific microorganism may be mixed and cultured in addition to the microorganism. That is, in the second agar medium, a microorganism group including symbiotic microorganisms, the aforementioned liquid culture microorganism group, and the like may be cultured.
なお「第一の寒天培地」及び「第二の寒天培地」に添加される成分については、寒天の濃度以外、前述の通りである。また、培養条件についても、前述の通り、培養する微生物群、特定の微生物、共生微生物等に合わせて、適宜調整することができる。さらに、図２に示すように、かかる共培養法において、適宜、「第三の寒天培地」を用いてもよい。「第三の寒天培地」は、第一又は第二の寒天培地中の微生物の培養（生存・増殖）に必要な物質を豊富に含有させることにより、少なくとも栄養層としての役割を担わせることができる。 The components added to the “first agar medium” and the “second agar medium” are as described above except for the agar concentration. Further, as described above, culture conditions can be appropriately adjusted according to the group of microorganisms to be cultured, a specific microorganism, a symbiotic microorganism, and the like. Furthermore, as shown in FIG. 2, in such a co-culture method, a "third agar medium" may be used as appropriate. The "third agar medium" can at least play a role as a vegetative layer by abundantly containing a substance necessary for culture (survival and growth) of the microorganism in the first or second agar medium. it can.
以上、説明したように、前記フィルターを介することにより、他の微生物と混在させることなく、第一の寒天培地にて培養されている特定の微生物は、第二の寒天培地にて培養されている共生微生物から、生存・増殖に必要な因子の提供を受けることができる。そのため、かかる共培養法によれば、特定の微生物を効率良く分離して培養することができる。 As described above, the specific microorganism cultured in the first agar medium is cultured in the second agar medium without being mixed with other microorganisms by interposing the filter. The symbiotic microorganism can provide the factor necessary for survival and growth. Therefore, according to such a co-culture method, specific microorganisms can be efficiently separated and cultured.
＜微生物の生産方法＞
前述の通り、公知の分離培養（例えば、特定の抗生物質を添加した寒天培地を用いた塗抹培養）においては培養することのできない非優占的な微生物を増殖させることができる。従って、本発明は、非優占的な微生物（特定の微生物）を生産するための下記方法を提供することもできる。
<Method of producing microorganisms>
As described above, non-dominant microorganisms which can not be cultured can be grown in known separation culture (for example, smear culture using an agar medium to which a specific antibiotic is added). Therefore, the present invention can also provide the following method for producing non-dominant microorganisms (specific microorganisms).
前述の方法によって、微生物群から前記特定の微生物を分離して培養する工程と、
前記工程にて分離培養した前記特定の微生物を回収する工程と
を含む方法。
Separating and culturing the specific microorganism from the group of microorganisms by the method described above;
Recovering the specific microorganism separated and cultured in the step.
かかる工程において、特定の微生物を回収する方法としては特に制限はなく、特定の微生物の性質に合わせて、公知の方法を適宜選択することができる。また、かかる特定の微生物を生産するための方法は、特定の微生物そのものの生産のみならず、該微生物から産生される有用な物質の生産方法としても好適に用いることができる。 There is no particular limitation on the method of recovering a specific microorganism in such a step, and a known method can be appropriately selected according to the nature of the specific microorganism. Moreover, the method for producing such a specific microorganism can be suitably used not only as the production of the specific microorganism itself but also as a method of producing useful substances produced from the microorganism.
＜新規な微生物のスクリーニング方法＞
前述の通り、公知の分離培養においては培養することのできない非優占的な微生物を培養することができる。さらに後述の実施例において示す通り、このようして得られた非優占的な微生物の中から、新規な微生物を同定することもできる。従って、本発明は、微生物群から新規な微生物をスクリーニングするための下記方法を提供することもできる。
<Method of screening novel microorganisms>
As mentioned above, non-dominant microorganisms which can not be cultured in known separation culture can be cultured. Further, as shown in the examples below, novel microorganisms can also be identified from thus obtained non-dominant microorganisms. Therefore, the present invention can also provide the following method for screening novel microorganisms from the microorganism group.
前述の方法によって、微生物群から前記特定の微生物を分離して培養する工程と、
前記工程にて分離培養した前記特定の微生物の特性を分析する工程と、
前記工程にて得られた特性と公知の微生物の特性とを比較し、一致しない場合には、前記特定の微生物は新規な微生物であると判定する工程と
を含む方法。
Separating and culturing the specific microorganism from the group of microorganisms by the method described above;
Analyzing the characteristics of the specific microorganism separated and cultured in the step;
And D. comparing the property obtained in the step with the property of a known microorganism, and if not identical, determining that the specific microorganism is a novel microorganism.
本発明にかかるスクリーニング方法において、分析対象となる「特性」としては、他の微生物と相違する性質であれば特に制限はなく、例えば、ゲノムＤＮＡ、ｍＲＮＡ又はタンパク質等の配列情報が挙げられる。かかる特性を分析する手段としても特に制限はなく、例えば、マイクロアレイによる分析、全ゲノムショットガン法、１６Ｓ ｒＲＮＡ遺伝子の配列決定法が挙げられる。 In the screening method according to the present invention, the “characteristics” to be analyzed is not particularly limited as long as it is a property different from other microorganisms, and examples thereof include sequence information such as genomic DNA, mRNA or protein. The means for analyzing such characteristics are not particularly limited, and examples thereof include analysis by microarray, whole genome shotgun method, and sequencing method of 16S rRNA gene.
また、かかる分析により得られた特性を、公知の微生物の特性と比較する方法についても特に制限はなく、当業者であれば、各特性に適した比較方法及びその基準に則して、公知の微生物の特性との一致又は不一致を判断することができる。 Also, there is no particular limitation on a method of comparing the characteristics obtained by such analysis with the characteristics of known microorganisms, and those skilled in the art will be known in accordance with the comparison method suitable for each characteristic and its standard. A match or mismatch with the characteristics of the microorganism can be determined.
例えば、特性が１６Ｓ ｒＲＮＡ遺伝子の配列情報である場合には、特定の微生物の１６Ｓ ｒＲＮＡ遺伝子の塩基配列について、公知の微生物の１６Ｓ ｒＲＮＡ遺伝子の配列情報が登録されているＧｅｎｂａｎｋ等のデータベースに対し、ＢＬＡＳＴによる相同性検索（ｈｔｔｐ：／／ｂｌａｓｔ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／Ｂｌａｓｔ．ｃｇｉ？ＣＭＤ＝Ｗｅｂ＆ＰＡＧＥ＿ＴＹＰＥ＝ＢｌａｓｔＨｏｍｅ）を行うことにより、特定の微生物の特性と公知の微生物の特性とを比較することができる。そして、その結果、特定の微生物の１６Ｓ ｒＲＮＡ遺伝子の塩基配列に対する相同性が９８．７％以上である公知の微生物が、前記データベースに登録されていなければ、特定の微生物と公知の微生物とは一致しておらず、当該特定の微生物は新規な微生物であると判定することができる。 For example, when the characteristic is the sequence information of the 16S rRNA gene, with respect to the base sequence of the 16S rRNA gene of a specific microorganism, the database such as Genbank in which the sequence information of the 16S rRNA gene of a known microorganism is registered. Compare the characteristics of a specific microorganism with the characteristics of a known microorganism by performing homology search by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome) be able to. And as a result, if a known microorganism having 98.7% or more homology to the nucleotide sequence of the 16S rRNA gene of a specific microorganism is not registered in the database, the specific microorganism and the known microorganism are one. The particular microorganism can be determined to be a novel microorganism.
以下、実施例に基づいて本発明をより具体的に説明するが、本発明は以下の実施例に限定されるものではない。また、本実施例は、下記の通りに調製した試料、培地及び抗生物質を用いて行った。 Hereinafter, the present invention will be more specifically described based on examples, but the present invention is not limited to the following examples. Moreover, this example was performed using the sample, the culture medium, and the antibiotic which were prepared as follows.
＜腸内常在菌混合懸濁液＞
糞便は、各被験者（ヒト）から採取した後、速やかに冷蔵にて保管したものを使用した。そして、各新鮮糞便を希釈液Ａにて１０２倍希釈することにより、腸内常在菌混合懸濁液として調製した。希釈液Ａの組成を表１に示す。
<Intestinal indigenous bacteria mixed suspension>
The feces were collected from each subject (human) and stored immediately under refrigeration. Then, each fresh feces was diluted 10 2 times with diluent A to prepare a mixed suspension of intestinal resident bacteria. The composition of Dilution A is shown in Table 1.
＜ＮＴ培地＞
腸内常在菌混合懸濁液を液体培養するために用いたＮＴ液体培地、及び、塗抹培養（分離培養）等するために用いたＮＴ寒天培地の組成を表２に示す。また、ＮＴ液体培地の成分である「５×ソルト」の組成を表３に示す。
<NT medium>
Table 2 shows the composition of the NT liquid medium used for liquid culture of the enteric indigenous bacterial mixed suspension and the NT agar medium used for smear culture (separate culture) and the like. Also, the composition of “5 × salt”, which is a component of the NT liquid medium, is shown in Table 3.
＜抗生物質＞
前記ＮＴ液体培地には、表４に記載の抗生物質を、単独又は組み合わせて添加して用いた。
<Antibiotics>
The antibiotics described in Table 4 were used alone or in combination in the NT liquid medium.
図１に示す通り、腸内常在菌混合懸濁液（図中「微生物群」）中の非優占的な微生物（図中「菌Ｃ」及び「菌Ｄ」）を培養するためには、増殖速度の速い優占的な微生物（図中「菌Ａ」及び「菌Ｂ」）の対数増殖期に、抗生物質にて処理しながら培養することが有効ではないかと想定し、下記培養を行った。 As shown in FIG. 1, in order to cultivate non-dominant microorganisms (“fungus C” and “fungus D” in the figure) in a mixed suspension of enteric indigenous bacteria (“microbe group” in the figure) In the logarithmic growth phase of dominant microorganisms with fast growth rate (“fungus A” and “fungus B” in the figure), it is assumed that it is effective to culture while treating with antibiotics, went.
すなわち、先ず、腸内常在菌混合懸濁液をＮＴ液体培地に１／１００量接種し、３７℃、ＣＯ２ガス下にて嫌気培養を行った。また、この嫌気培養は、図３に示す通り、ノルフロキサシン、ホスホマイシン、テトラサイクリン若しくはトリメトプリム、又は、前記４種の抗生物質の存在下、培養開始〜７時間後迄（図中「１」の条件）、培養開始〜５．５時間後迄（図中「２」の条件）又は培養開始してから４〜７時間迄（図中「３」の条件）行った。 That is, first, 1/100 amount of the mixed liquid of enteric indigenous bacteria was inoculated to NT liquid medium, and anaerobic culture was performed at 37 ° C. under CO 2 gas. In addition, as shown in FIG. 3, this anaerobic culture is performed in the presence of norfloxacin, fosfomycin, tetracycline or trimethoprim, or the above four antibiotics for 7 hours after the initiation of culture (condition of "1" in the figure), From the start of culture to 5.5 hours later (the condition of "2" in the figure) or 4 to 7 hours after the start of culture (the condition of "3" in the figure).
なお、抗生物質存在下における培養時間は、腸内常在菌混合懸濁液をＮＴ液体培地に１／１００量接種し、３７℃、ＣＯ２ガス下、抗生物質無添加にて嫌気培養を行った際の対数増殖期（嫌気培養を開始してから４〜８時間迄）が、増殖速度の速い優占的な微生物の対数増殖期に相当するものとして設定した（図４参照）。 In addition, culture time in the presence of antibiotics inoculates 1/100 amount of the intestinal suspension mixed bacterial suspension into NT liquid medium and performs anaerobic culture without addition of antibiotics under 37 ° C., CO 2 gas. The log growth phase (up to 4 to 8 hours after the start of anaerobic culture) at the time of growth was set as that corresponding to the log growth phase of the dominant microorganism having a high growth rate (see FIG. 4).
前記の通り、図３に記載の条件にて嫌気培養を行った後、遠心分離（１００００ｒｐｍ，１０分，１０℃）を行い、添加した抗生物質が含まれる上清部分を取り除いた。得られた沈殿（菌体）を各々、希釈液Ａにて１０２〜１０４倍希釈した。次いで、各希釈液１００μＬをＮＴ寒天培地に塗抹し、嫌気培養を行った。培養７日後に寒天培地上の一定範囲に認められるコロニーを無作為に釣菌し、継代培養を行った。そして、継代培養７日後に、コロニーの様子を観察した。また、常法に従って、これらコロニーからゲノムＤＮＡを抽出し、１６ＳｒＲＮＡ遺伝子の配列を決定し、菌種の同定を行った。 As described above, after anaerobic culture was performed under the conditions described in FIG. 3, centrifugation (10000 rpm, 10 minutes, 10 ° C.) was performed to remove the supernatant portion containing the added antibiotic. The resulting precipitates (cells) were each diluted 10 2 to 10 4 times with diluent A. Then, 100 μL of each dilution was smeared on NT agar medium and anaerobic culture was performed. After 7 days of culture, colonies found in a certain range on the agar medium were picked at random and subcultured. Then, after 7 days of subculture, the appearance of colonies was observed. Moreover, genomic DNA was extracted from these colonies according to a conventional method, the sequence of the 16S rRNA gene was determined, and the bacterial species were identified.
また、腸内常在菌混合懸濁液を、ＮＴ液体培地に１／１００量接種し、３７℃、ＣＯ２ガス下、抗生物質非存在下にて嫌気培養を行った。そして、前記抗生物質存在下における培養同様に、遠心分離を行い、得られた沈殿を各々、希釈液Ａにて１０２〜１０４倍希釈した。次いで、各希釈液１００μＬをＮＴ寒天培地に塗抹し、７日間嫌気培養した。そして、得られたコロニーの１６ＳｒＲＮＡ遺伝子の配列を決定し、菌種の同定を行った。 In addition, 1/100 volume of NT suspension was inoculated into NT liquid medium mixed suspension, and anaerobic culture was performed at 37 ° C. under CO 2 gas in the absence of antibiotics. Then, centrifugation was performed in the same manner as in the culture in the presence of the antibiotic, and the obtained precipitates were each diluted 10 2 to 10 4 times with Diluent A. Then, 100 μL of each dilution was smeared on NT agar medium and anaerobically cultured for 7 days. Then, the sequence of the 16S rRNA gene of the obtained colony was determined to identify the bacterial species.
さらに、新鮮糞便サンプルを希釈液Ａにて１０７倍希釈したものを、ＮＴ寒天培地に塗抹し、嫌気培養した。そして、その７日後、得られたコロニーの１６ＳｒＲＮＡ遺伝子の配列を決定し、菌種の同定を行った。 Furthermore, fresh feces samples diluted 10 7 times with Dilution A were smeared on NT agar medium and anaerobically cultured. Then, seven days later, the sequence of the 16S rRNA gene of the obtained colony was determined to identify the bacterial species.
以上の実験につき、得られた結果を表５〜１２及び図５〜１０に示す。なお、図５〜９は、表５〜９に示した結果を各々円グラフにて表わしたものである。図９及び１０において、四角枠で囲まれている菌株は、今回新たに同定された新規菌株であることを示す。 The obtained result is shown to Tables 5-12 and FIGS. 5-10 about the above experiment. 5 to 9 show the results shown in Tables 5 to 9 in a circle graph, respectively. In FIGS. 9 and 10, the strain surrounded by a square frame indicates that it is a novel strain newly identified this time.
表５〜８及び図５〜８に示した結果から明らかなように、単種の抗生物質存在下における液体培養においては、特定の菌株による占有率が大きく、特定の３種の菌株が、検査したコロニー全体の７０％以上を占有していた。 As apparent from the results shown in Tables 5-8 and FIGS. 5-8, in liquid culture in the presence of a single type of antibiotic, the occupancy rate by a particular strain is large, and the particular 3 strains are tested. Occupied 70% or more of the entire colony.
一方、表９及び図９に示した結果から明らかなように、これらの抗生物質４種存在下にて液体培養した場合には、前記単種の抗生物質存在下における液体培養とは異なり、数種の特定の菌株によって優占的にコロニーが形成されてしまうことはなかった。具体的には、単種の抗生物質存在下においては、特定の３種の菌株による占有率が７０％以上であったが、抗生物質４種を併用して培養した場合には、５５％弱に抑えられていた。また、単種の抗生物質存在下の培養においては、検出されなかった多数の菌株を、この抗生物質併用培養により検出することが可能となった。さらに、かかる培養方法によれば、抗生物質非存在下にて液体培養した場合（表１０ 参照）や、液体培養せずに腸内常在菌混合懸濁液を直接塗抹培養した際（表１１ 参照）には認められない菌種を、多く検出できることが明らかになった。また、図１０に示す通り、前記抗生物質４種存在下にて液体培養した場合には、今まで同定されていなかった新規な菌を、腸内常在菌群から分離して培養することができ、それらの存在を検出できることも明らかになった。 On the other hand, as is clear from the results shown in Table 9 and FIG. 9, when liquid culture is performed in the presence of four types of these antibiotics, the number is different from liquid culture in the presence of the single type of antibiotic, Colonies were not formed dominantly by certain strains of the species. Specifically, in the presence of a single type of antibiotic, the occupancy rate by specific three strains was 70% or more, but when it is cultured in combination with 4 types of antibiotics, it is less than 55% It was suppressed by In addition, in the culture in the presence of a single type of antibiotic, it became possible to detect many strains not detected by this antibiotic combined culture. Furthermore, according to this culture method, when liquid culture is carried out in the absence of antibiotics (see Table 10), or when direct smear culture is carried out on the intestinal resident mixture mixed suspension without liquid culture (Table 11) It became clear that many bacterial species not found in (see) can be detected. In addition, as shown in FIG. 10, when liquid culture is performed in the presence of the four antibiotics, a novel bacterium which has not been identified until now may be isolated from the intestinal resident bacteria group and cultured. It also became clear that they could and could detect their presence.
また、表１２に示した結果から明らかなように、複数の抗生物質にて培養開始直後から７時間迄処理した場合には、培養開始後４〜７時間迄処理した場合には認められない菌属（Ｄｅｓｕｌｆｏｔｏｍａｃｕｌｕｍ属，Ｈｏｌｅｍａｎｉａ属，Ｏｄｏｎｉｂａｃｔｅｒ属，Ａｋｋｅｒｍａｎｓｉａ属）が分離された。さらにまた、複数の抗生物質にて培養開始直後から５．５時間後迄処理した場合においても、ＢＬ１１８３、Ｌａｃｔｏｂａｃｉｌｌｕｓ属、Ｄｏｒｅａ属といった、培養開始後４〜７時間迄処理した場合には認められない菌種／菌属が分離された。このように、複数の抗生物質による処理時期及びその時間の違いによっても、分離される菌株に違いが認められることが明らかとなった。 Also, as is clear from the results shown in Table 12, when treated with multiple antibiotics for 7 hours immediately after the start of culture, bacteria not found when treated for 4 to 7 hours after start of culture The genera (Desulfotomaculum, Holemania, Odonibacter, Akkermansia) was isolated. Furthermore, even when treated with multiple antibiotics for 5.5 hours immediately after the start of culture, no treatment is observed when treated for 4 to 7 hours after the start of culture, such as BL1183, Lactobacillus, and Dorea. Bacterial species / genus were isolated. Thus, it was revealed that differences in the strains to be separated were also recognized by differences in the time and time of treatment with multiple antibiotics.
次に、今回分離培養することができ、新規に同定された菌株の一部について、各抗生物質に対する最小発育阻止濃度（ＭＩＣ）を調べた。すなわち、−８０℃に保存した菌株をＮＴ液体培地に接種して２日間培養したものを、新しい同液体培地に１／１０接種して１日間継代培養した。その後、種々の濃度の各抗生物質（ノルフロキササシン，テトラサイクリン，トリメトプリム，ホスホマイシン）を各々添加したＮＴ寒天培地上にスポットし、嫌気培養した。そして、その５日後に、コロニー形成の様子を観察してＭＩＣを判定した。得られた結果を表１３に示す。 Next, the minimum inhibitory concentration (MIC) for each antibiotic was examined for some of the newly identified strains that could be isolated and cultured this time. That is, the strain stored at -80 ° C was inoculated into NT liquid medium and cultured for 2 days, and then 1/10 of the same liquid medium was inoculated into fresh liquid medium and subcultured for 1 day. Thereafter, the cells were spotted on NT agar medium to which various antibiotics (norfloxasacin, tetracycline, trimethoprim, fosfomycin) of various concentrations were respectively added, and anaerobically cultured. Then, five days later, the appearance of colony formation was observed to determine MIC. The obtained results are shown in Table 13.
表１３に示した結果から明らかなように、今回新規に同定された菌株は、いずれも前記液体培養時に添加された抗生物質に対して耐性を有しておらず、また、それら菌株のＭＩＣは、前記液体培養時に添加された抗生物質の濃度以下であることが明らかになった。従って、今回新規に同定された菌株に関しては、対数増殖期が抗生物質処理期間よりも遅かったため、分離培養できたことが示唆される。 As is clear from the results shown in Table 13, none of the strains newly identified at this time have resistance to the antibiotics added during the liquid culture, and the MICs of these strains are , It was revealed that the concentration was less than the concentration of antibiotics added at the time of the liquid culture. Therefore, with respect to the strain newly identified this time, the log growth phase was later than the antibiotic treatment period, suggesting that separate culture could be performed.
次に、複数の被験者の腸内常在菌混合懸濁液を対象にして、前記同様に、培養開始してから４〜７時間迄、複数の抗生物質の存在下にて液体培養した後、塗抹培養を行い、得られたコロニーの１６ＳｒＲＮＡ遺伝子の配列を決定し、菌種の同定を行った。得られた結果を表１４に示す。 Next, after subjecting the mixed suspension of enteric indigenous bacteria of a plurality of test subjects to liquid culture in the presence of a plurality of antibiotics for 4 to 7 hours after the start of culture as described above, The smear culture was performed, the sequence of the 16S rRNA gene of the obtained colony was determined, and the bacterial species were identified. The obtained results are shown in Table 14.
表１４に示した結果から明らかな通り、いずれの被験者から検出された菌株も前記同様に、その大半は、通常の塗抹培養では認められにくい菌種が多くを占め、その中には新規菌株（ＢＬ８２６、ｕｎｉｄｅｎｔｉｆｉｅｄ ｂａｃｔｅｒｉａ ＣＪ７、ＢＬ１８３２、、ＢＬ１８７９）も複数含まれていた。また分離された菌株を被験者間で比較すると、菌種レベルで個人ごとに多様性が認められた。 As is clear from the results shown in Table 14, as for the strains detected from any of the subjects, most of them are those which are difficult to be recognized in normal smear culture, and among them, novel strains A plurality of BL826, unidentified bacteria CJ7, BL1832, and BL1879 were also included. Moreover, when the isolate | separated strain was compared between test subjects, the diversity was recognized for each individual | organisms in the microbe level.
従って、本発明の方法を用いることにより、様々なヒトの腸内常在菌等をスクリーニングすることにより、未同定菌株を含む非優占菌の多様性が観察できることが明らかになった。 Therefore, it has become clear that the diversity of non-dominant bacteria, including unidentified strains, can be observed by screening various human intestinal indigenous bacteria and the like by using the method of the present invention.
以上説明したように、本発明によれば、非優占的な微生物を培養することが可能となる。したがって、本発明は、非優占的な微生物に含まれている可能性が非常に高い、新規な有用微生物のスクリーニング方法、及びそれら微生物の生産方法等として有用である。 As described above, according to the present invention, it is possible to culture non-dominant microorganisms. Therefore, the present invention is useful as a novel screening method of useful microorganisms, which is very likely to be contained in non-dominant microorganisms, a method of producing these microorganisms, and the like.
１…第一の寒天培地、２…第二の寒天培地、３…第三の寒天培地、４…フィルター、５…スぺーサー、６…培養容器。 1 ... 1st agar medium, 2 ... 2nd agar medium, 3 ... 3rd agar medium, 4 ... filter, 5 ... spacer, 6 ... culture vessel.
Claims (6)
前記微生物群が抗生物質非存在下にて対数増殖する期間の一部又は全部において、前記微生物群を抗生物質存在下にて液体培養する工程と、
前記工程にて液体培養した微生物群を、前記抗生物質非存在下にて分離培養する工程とを含み、
かつ、前記抗生物質が、核酸合成阻害剤、タンパク質合成阻害剤、細胞壁合成阻害剤及び葉酸合成阻害剤の組み合わせである方法。 A method for separating and culturing a specific microorganism from a microorganism group,
Liquid culturing the microorganism group in the presence of an antibiotic during part or all of a period during which the microorganism group grows logarithmically in the absence of the antibiotic;
Separating and culturing the microorganism group liquid-cultured in the step, in the absence of the antibiotic,
And, the method wherein the antibiotic is a combination of a nucleic acid synthesis inhibitor, a protein synthesis inhibitor, a cell wall synthesis inhibitor and a folate synthesis inhibitor .
請求項１〜４のうちのいずれか１項に記載の方法によって、微生物群から前記特定の微生物を分離して培養する工程と、
前記工程にて分離培養した前記特定の微生物を回収する工程と
を含む方法。 A method for producing a specific microorganism,
Separating and culturing the specific microorganism from the group of microorganisms by the method according to any one of claims 1 to 4 ;
Recovering the specific microorganism separated and cultured in the step.
請求項１〜４のうちのいずれか１項に記載の方法によって、微生物群から前記特定の微生物を分離して培養する工程と、
前記工程にて分離培養した前記特定の微生物の特性を分析する工程と、
前記工程にて得られた特性と公知の微生物の特性とを比較し、一致しない場合には、前記特定の微生物は新規な微生物であると判定する工程と
を含む方法。 A method for screening novel microorganisms from a microbial community comprising:
Separating and culturing the specific microorganism from the group of microorganisms by the method according to any one of claims 1 to 4 ;
Analyzing the characteristics of the specific microorganism separated and cultured in the step;
And D. comparing the property obtained in the step with the property of a known microorganism, and if not identical, determining that the specific microorganism is a novel microorganism.
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