JPH062049B2 - Legionella bacterium selective separation medium - Google Patents
Legionella bacterium selective separation mediumInfo
- Publication number
- JPH062049B2 JPH062049B2 JP59056572A JP5657284A JPH062049B2 JP H062049 B2 JPH062049 B2 JP H062049B2 JP 59056572 A JP59056572 A JP 59056572A JP 5657284 A JP5657284 A JP 5657284A JP H062049 B2 JPH062049 B2 JP H062049B2
- Authority
- JP
- Japan
- Prior art keywords
- medium
- legionella
- bacteria
- gvp
- amphotericin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
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Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は環境あるいは臨床材料からレジオネラ菌(Legio
nella spp.)を分離するためのレジオネラ菌選択分離用
培地に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application]
nella spp.) for separating Legionella bacteria selective separation medium.
レジオネラ菌は在郷軍人病（Legionnaire's disease）
の原因菌として注目されているが、その発育条件は一般
の細菌に比較して極めて厳しく、現在いわゆるＣＹＥ、
Ｂ−ＣＹＥおよびＦ−Ｇ培地等の限られた培地でしか発
育させることができない。たとえばＢ−ＣＹＥ培地はＡ
ＣＥＳ（Ｎ−２−アセトアミド−２−アミノエタンスル
ホン酸）とＫＯＨとの溶液に酵母エキス、活性炭末およ
び寒天を加え、さらにＬ−シスチンおよびピロリン酸第
二鉄を添加して調製される培地であり、レジオネラ菌の
発育およびその集落の観察に適した培地として知られて
いる（Pasculle,A.W.,et.al:J.Inf.Dis。141;727,1980)。
しかし、このＢ−ＣＹＥ培地又は前記ＣＹＥおよびＦ−
Ｇ培地ではレジオネラ菌の集落を形成させるまでに他の
細菌と異なつて３〜７日間の長時間の培養を必要とす
る。しかも環境あるいは臨床材料から得られる検体中で
はレジオネラ菌が単独で存在することはほとんどない。
したがつて前記酵母エキス等の発育因子のみで構成され
ているこのＢ−ＣＹＥ培地による分離試験では、培養中
にレジオネラ菌以外の細菌がより早く発育してそれらの
集落が培地表面全体を覆つてしまうため、レジオネラ菌
の検出は実質的には不可能であつた。Legionella bacteria are Legionnaire's disease
It is attracting attention as a causative bacterium, but its growth conditions are extremely strict compared with general bacteria, and so-called CYE,
It can only be grown in limited media such as B-CYE and FG media. For example, B-CYE medium is A
In a medium prepared by adding yeast extract, activated carbon powder and agar to a solution of CES (N-2-acetamido-2-aminoethanesulfonic acid) and KOH, and further adding L-cystine and ferric pyrophosphate. It is known as a medium suitable for the growth of Legionella and observation of its colonies (Pasculle, AW, et.al: J. Inf. Dis. 141; 727,1980).
However, this B-CYE medium or the CYE and F-
Unlike the other bacteria, G medium requires long-term culture for 3 to 7 days before forming a colony of Legionella. Moreover, Legionella bacteria rarely exist alone in samples obtained from the environment or clinical materials.
Therefore, in the separation test using the B-CYE medium composed only of the growth factors such as the yeast extract, bacteria other than Legionella grow faster during the culture, and their colonies cover the entire surface of the medium. Therefore, it was virtually impossible to detect Legionella.
このためＢ−ＣＹＥ培地にグリシン、バンコマイシンお
よびポリミキシンＢを添加してレジオネラ菌に対する選
択性を持たせた培地（ＧＶＰ培地）がワドウスキイ（Wa
dowsky）およびイー（Yee）によつて提案されており
（１９８１年）、このＧＶＰ培地ではレジオネラ菌以外
の細菌の発育を阻害することが可能である。しかし、こ
の培地は真菌の発育をほとんど阻止しないので、検体中
に真菌特に糸状菌が存在する場合には、一般の細菌の集
落の形成が抑止されていることも相加的に作用して培地
表面全体に菌糸が発育し、レジオネラ菌の検出を著しく
困難にする。For this reason, a medium (GVP medium) in which glycine, vancomycin and polymyxin B are added to B-CYE medium so as to have selectivity for Legionella bacteria is Wadowski (Wa
Proposed by Dowsky and Yee (1981), this GVP medium is capable of inhibiting the growth of bacteria other than Legionella. However, since this medium hardly inhibits the growth of fungi, when fungi, especially filamentous fungi, are present in the sample, the fact that the formation of colonies of general bacteria is suppressed also acts additively. Mycelia grow on the entire surface, making detection of Legionella bacteria extremely difficult.
本発明の目的は前記従来技術における問題点を解消し、
従来培地の選択性をさらに向上させてレジオネラ菌の選
択分離能にすぐれた培地を提供することにある。The object of the present invention is to solve the problems in the prior art,
Another object of the present invention is to provide a medium excellent in the selective separation ability of Legionella bacteria by further improving the selectivity of the conventional medium.
本発明者等は前記目的の達成のためにレジオネラ菌の培
養中における一般細菌および真菌の発生を極力抑止する
ことについて研究を重ねた結果、ＧＶＰ培地に対してア
ムホテリシンＢを特定の範囲の添加量で加えた培地がレ
ジオネラ菌の選択的な検出に極めて有効であることを発
見して本発明を完成するに到つた。In order to achieve the above-mentioned object, the inventors of the present invention have conducted extensive research on suppressing the development of general bacteria and fungi during the culture of Legionella bacteria, and as a result, added amphotericin B to the GVP medium within a specific range. The present invention was completed by discovering that the medium added in 1. is extremely effective for selective detection of Legionella.
すなわち、本発明はＢ−ＣＹＥ培地にグリシン、バンコ
マイシンおよびポリミキシンＢを含有させてなるレジオ
ネラ菌選択分離用培地（ＧＶＰ培地）に対し、該ＧＶＰ
培地１について40〜800mgのアムホテリシンＢを添加
したことを特徴とする。That is, the present invention relates to a GVP medium for selective separation of Legionella bacteria (GVP medium), which comprises B-CYE medium containing glycine, vancomycin and polymyxin B.
It is characterized in that 40 to 800 mg of amphotericin B was added to the medium 1.
前記ＧＶＰ培地に各種抗真菌剤を添加して真菌の発育を
抑止しながらレジオネラ菌のみを選択的に検出する際に
はアムホテリシンＢがとりわけすぐれた選択分離能を示
す。すなわち、レジオネラ菌に対する抗真菌剤の選択分
離能を確認するために、入手可能なレジオネラ菌の中か
ら任意に選択されたレジオネラニューモフィラ（Legion
ella pneumophila）9134および9137の保存株を、Ｂ−Ｃ
ＹＥ培地、ＧＶＰ培地およびＧＶＰ培地に各種抗真菌剤
を添加した培地に対して接種し、48時間培養した際の出
現数（cells/ml）は次表１の通りである。Amphotericin B exhibits a particularly excellent selective separation ability when selectively detecting only Legionella bacteria while inhibiting the growth of fungi by adding various antifungal agents to the GVP medium. That is, in order to confirm the selective separation ability of an antifungal agent against Legionella bacteria, Legionella pneumophila (Legionella) arbitrarily selected from the available Legionella bacteria is confirmed.
ella pneumophila) 9134 and 9137 conserved strains
Table 1 shows the number of cells (ml / ml) that appeared when the cells were inoculated into YE medium, GVP medium and GVP medium to which various antifungal agents had been added and cultured for 48 hours.
すなわち、本発明において用いられるアムホテリシンＢ
はＧＶＰ培地に対して20〜160mg/の範囲の添加量では
そのレジオネラ菌に対する抗菌作用がＧＶＰ培地自体の
場合ほとんど変りがない。これに対して同様な抗真菌剤
であるミコナゾールでは40mg/の添加量でレジオネラ
菌の出現数が減少し80mg/の添加量ではレジオネラ菌
が検出されなくなる。その他の抗真菌剤としてのシクロ
ヘキシミド、アニソマイシンおよびナイスタチンはこの
試験ではアムホテリシンＢと同様なレジオネラ抗菌作用
を示しているが、これらは後に述べる理由で必らずしも
本発明の目的には適合しない。 That is, the amphotericin B used in the present invention
When the amount of GVP medium added is in the range of 20 to 160 mg / g, the antibacterial action against Legionella bacteria is almost unchanged in the case of GVP medium itself. On the other hand, with miconazole, which is a similar antifungal agent, the appearance number of Legionella bacteria decreases at the addition amount of 40 mg /, and Legionella bacteria becomes undetectable at the addition amount of 80 mg /. Cycloheximide, anisomycin and nystatin as other antifungal agents show legionella antibacterial activity similar to amphotericin B in this test, but they are not always suitable for the purpose of the present invention for the reason described below. .
さらに、アムホテリシンＢはそのレジオネラ菌および真
菌に対するＭＩＣ（最小発育阻止濃度）の差が大きい。
たとえば入手可能なレジオネラ菌の中から任意に選択し
た表２に示すレジオネラニューモフィラ（Legionella p
neumophila）9134以下のレジオネラ保存株をＢ−ＣＹＥ
培地に接種した際のアムホテリシンＢのこれらレジオネ
ラ菌の各菌株に対するＭＩＣを表２に示す。ＧＶＰ培地
を基準として約1600μｇ／mlであるのに対しカンジダ属
（Candida albicans CAND−１等）に対するＭＩＣは12.
5μｇ／mlである。したがつて、ＧＶＰ培地に対してア
ムホテリシンＢを前記範囲内の量で添加すればレジオネ
ラ菌が他の細菌から分離選択されると共に、特に前記培
地中における真菌の発育を抑止してレジオネラ菌の検出
率を高めることができる。本発明の培地におけるアムホ
テリシンＢの特に効果的な添加量の範囲は後述する実施
例の結果等に基いてＧＶＰ培地１について約40mg/
〜800mg/であり、特に約80mg/付近である。Furthermore, amphotericin B has a large difference in its MIC (minimum inhibitory concentration) against Legionella and fungi.
For example, Legionella pneumophila (Legionella p
neumophila) 9134 or less conserved strains of Legionella B-CYE
The MICs of amphotericin B for each of these Legionella strains when inoculated into the medium are shown in Table 2. It is about 1600 μg / ml based on GVP medium, whereas the MIC for Candida (Candida albicans CAND-1 etc.) is 12.
5 μg / ml. Therefore, when Amphotericin B is added to the GVP medium in an amount within the above range, the Legionella bacterium is separated and selected from other bacteria, and particularly, the growth of the fungus in the medium is suppressed and the detection of Legionella bacterium is detected. The rate can be increased. The range of the particularly effective addition amount of amphotericin B in the medium of the present invention is about 40 mg / GVP medium 1 based on the results of Examples described later.
~ 800 mg /, especially about 80 mg /.
一方比較の為に用いたシクロヘキシミドでは表２に示す
ようにレジオネラ菌および真菌に対するＭＩＣの差が明
確ではなく、しかもその値が菌株によつてばらつきを示
す為にレジオネラ菌の選択分離を目的とする本発明につ
いては実用上アムホテリシンＢほど効果的ではない。On the other hand, in cycloheximide used for comparison, as shown in Table 2, the difference in MIC between Legionella bacterium and fungus is not clear, and since the value varies depending on the strain, the purpose is to selectively separate Legionella bacterium. The present invention is not as effective as amphotericin B in practice.
明細書第４頁第１８行および１９行、第５頁表１、およ
び第８頁表２中の各レジオネラ菌の菌株表示 （Ｌ．ニューモフィラ９１３４〜Ｌ．ジョーダニス３１
９３）は表２中に夫々併記された American type Cult
ure Collectionの寄託番号ＡＴＣＣNO.に対応する。 Strain indication of each Legionella strain in the specification, page 4, lines 18 and 19, page 5, table 1 and page 8, table 2 (L. pneumophila 9134-L. Jordanis 31)
93) are American type Cults listed in Table 2.
Corresponds to the deposit number ATCC NO. of the ure Collection.
以下本発明を実施例によりさらに説明する。The present invention will be further described below with reference to examples.
〔実施例１〕 冷却塔の冷却水（福岡県北部地区）から採つた15件の材
料について本発明による培地を使用してレジオネラ菌の
検出実験を行なつた。[Example 1] With respect to 15 materials collected from cooling water of a cooling tower (Fukuoka prefecture northern area), a culture experiment according to the present invention was used to carry out an experiment for detecting Legionella bacteria.
各材料は遠心分離により約100倍の濃度に濃縮し、pH2.2
の緩衝液で処理した後、各処理材料の100μを下記表
３の組成の供試培地に夫々接種して35℃で７日間培養
し、レジオネラ菌の検出を試みた。Each material was concentrated to a concentration of about 100 times by centrifugation and pH 2.2.
After treatment with the buffer solution of 1., 100 μm of each treated material was inoculated into the test medium having the composition shown in Table 3 below and cultured at 35 ° C. for 7 days to try to detect Legionella.
前記表３中、Ｂ−ＣＹＥおよびＧＶＰ培地ならびにＧＶ
Ｐ＋シクロヘキシミド培地は夫々比較のために用いた培
地である。 In Table 3 above, B-CYE and GVP medium and GV
P + cycloheximide medium is the medium used for comparison.
前記環境材料について行なつたレジオネラ菌の検出実験
の結果を表４に示す。表中、Ｉは培地一つ当り50個以上
の一般細菌の集落が出現した材料を、IIは培地中に真菌
が出現した材料をそしてIIIはレジオネラ菌が検出でき
た材料を夫々材料数および全材料（15件）に対する百分
比で示したものである。Table 4 shows the results of the detection experiment of Legionella bacteria conducted on the above environmental materials. In the table, I is a material in which 50 or more colonies of general bacteria appear per medium, II is a material in which fungi appear in the medium, and III is a material in which Legionella bacteria can be detected. It is shown as a percentage of the material (15 cases).
前記表４が示すように、レジオネラ菌以外の一般細菌に
対する選択的な生育抑止物質を含まないＢ−ＣＹＥ培地
では、レジオネラ菌以外の細菌が全ての材料について培
地表面全体に発育してしまいまた一部の材料では真菌の
集落も出現してレジオネラ菌の検出は各材料とも不可能
であつた（検出率０％）。 As shown in Table 4, in the B-CYE medium containing no selective growth inhibitory substance against general bacteria other than Legionella bacteria, bacteria other than Legionella bacteria grew on the entire surface of the medium for all the materials. In the materials of the above parts, colonies of fungi also appeared and it was impossible to detect Legionella bacteria (detection rate 0%) in each material.
一方、選択的な生育物質としてのグリシン、バンコマイ
シンおよびポリミキシンＢを前記Ｂ−ＣＹＥ培地に添加
したＧＶＰ培地では、各材料についてレジオネラ菌以外
の細菌の発育は阻止されたが、代つて全ての材料につい
て真菌集落が出現し、このためにレジオネラ菌の分離は
極めて困難であつた（検出率約27％）。On the other hand, in GVP medium in which glycine, vancomycin and polymyxin B as selective growth substances were added to the B-CYE medium, the growth of bacteria other than Legionella was inhibited for each material, but instead for all materials Fungal colonies appeared, which made the isolation of Legionella bacteria extremely difficult (detection rate about 27%).
これに対して前記ＧＶＰ培地に対して抗真菌材としての
アムホテリシンＢを培地１について80mg添加してなる
本発明実施例の培地を用いた場合には、レジオネラ菌以
外の細菌の発育がＧＶＰ培地の場合と同様に阻止される
と共に、真菌集落の出現する材料の割合はＧＶＰ培地の
場合を100％として27％に減少した。しかも真菌集落の
形成された材料でもその成長がアムホテリシンＢによつ
て妨げられるので、レジオネラ菌の検出が極めて容易に
なり全ての材料についてレジオネラ菌の検出が可能であ
つた（検出率100％）。また、比較のためにシクロヘキ
シミドを加えたＧＶＰ培地では真菌集落の出現した材料
が全体の60％になり、またレジオネラ菌の検出率もアム
ホテリシンＢに比較し低下していた。On the other hand, when the medium of Example of the present invention in which 80 mg of Amphotericin B as an antifungal material was added to the GVP medium in regard to the medium 1, the growth of bacteria other than Legionella was observed in the GVP medium. In the same manner as in the case, the proportion of the material in which fungal colonies appeared was reduced to 27% with GVP medium as 100%. Moreover, since the growth of the material in which the fungal colony is formed is also hindered by the amphotericin B, the detection of Legionella bacterium becomes extremely easy and the detection of Legionella bacterium was possible for all the materials (detection rate 100%). For comparison, in the GVP medium containing cycloheximide, the material in which fungal colonies appeared was 60% of the whole, and the detection rate of Legionella bacteria was lower than that of amphotericin B.
〔実施例２〜４〕 前記実施例１においてＧＶＰ培地に添加するアムホテリ
シンＢの量を夫々20、40および160mg/とし実施例１と
同様にしてレジオネラ菌の検出を行なつた。結果を表５
に示す。尚、表中にはＧＶＰ培地にアムホテリシンＢ以
外の抗真菌剤としての前記ミクロヘキシミドならびにナ
イスタチン、ミコナゾールおよびアニソマイシンを夫々
添加して調製した培地による実験結果を比較のために示
してある。尚表５中のＩ、IIおよびIIIは前記表中の対
応する欄と同一の内容すなわち一般細菌が所定数以上出
現した材料、真菌が出現した材料およびレジオネラ菌が
検出できた材料の数を夫々百分比で示す。Examples 2 to 4 In the same manner as in Example 1, the amounts of amphotericin B added to the GVP medium were 20, 40 and 160 mg / ml, respectively, and Legionella bacteria were detected in the same manner as in Example 1. The results are shown in Table 5.
Shown in. In addition, in the table, the experimental results using a medium prepared by adding the above-mentioned microheximide as an antifungal agent other than amphotericin B and nystatin, miconazole and anisomycin to GVP medium are shown for comparison. Incidentally, I, II and III in Table 5 have the same contents as the corresponding columns in the table, that is, the number of materials in which general bacteria appeared in a predetermined number or more, the number of materials in which fungi appeared and the number of materials in which Legionella bacteria could be detected, respectively. Shown as a percentage.
前記表５に示す実施例２〜４の結果からも明らかなよう
に、アムホテリシンＢを本発明の範囲内の量でＧＶＰ培
地に添加してなる培地はいずれも環境材料中のレジオネ
ラ菌の検出にすぐれた効果を示した。 As is clear from the results of Examples 2 to 4 shown in Table 5 above, any medium obtained by adding Amphotericin B to the GVP medium in an amount within the range of the present invention can detect Legionella bacteria in environmental materials. It showed an excellent effect.
これに対して、比較のために抗真菌剤としてシクロヘキ
シミドを用いた場合ではレジオネラ菌の検出率がアムホ
テリシンＢに比較して劣つている。またナイスタチンお
よびミコナゾールでは真菌の出現率が増加してレジオネ
ラ菌の検出が困難になる。アニソマイシンは真菌の出現
率が低くかつレジオネラ菌の検出率も比較的すぐれてい
るが、現在その入手が極めて困難でかつ高価な為実際の
使用および経済的見地からみて産業上の利用性にかけ
る。In contrast, when cycloheximide is used as an antifungal agent for comparison, the detection rate of Legionella is inferior to that of amphotericin B. In addition, nystatin and miconazole increase the incidence of fungi, making it difficult to detect Legionella. Although anisomycin has a low fungal appearance rate and a relatively good detection rate for Legionella, it is currently extremely difficult and expensive to obtain, and its practical use and economic applicability impair its utility. .
Claims (1)
ンおよびポリミキシンＢを含有させてなるレジオネラ菌
選択分離用培地（ＧＶＰ培地）に対し、該ＧＶＰ培地１
について40〜800mgのアムホテリシンＢを添加したこ
とを特徴とするレジオネラ菌選択分離用培地。1. A GVP medium 1 for a Legionella bacterium selective separation medium (GVP medium) comprising B-CYE medium containing glycine, vancomycin and polymyxin B.
About 40 to 800 mg of amphotericin B was added to the medium for selective separation of Legionella bacteria.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59056572A JPH062049B2 (en) | 1984-03-24 | 1984-03-24 | Legionella bacterium selective separation medium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59056572A JPH062049B2 (en) | 1984-03-24 | 1984-03-24 | Legionella bacterium selective separation medium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS60199398A JPS60199398A (en) | 1985-10-08 |
| JPH062049B2 true JPH062049B2 (en) | 1994-01-12 |
Family
ID=13030856
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP59056572A Expired - Fee Related JPH062049B2 (en) | 1984-03-24 | 1984-03-24 | Legionella bacterium selective separation medium |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH062049B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2739867B1 (en) * | 1995-10-13 | 1997-12-05 | Pasteur Sanofi Diagnostics | SELECTIVE MEDIA FOR CULTURE AND ISOLATION OF GRAM- BACTERIA, ANTIBIOTIC COMPOSITION |
| JP4632839B2 (en) * | 2005-03-31 | 2011-02-16 | アクアス株式会社 | Legionella selective culture medium and Legionella culturing method |
| SK287315B6 (en) | 2006-06-02 | 2010-06-07 | Biotika, A. S. | A method for polymyxin B isolation from fermented soil |
| SK287293B6 (en) | 2006-06-15 | 2010-05-07 | Biotika, A. S. | A method for fermentation of polymyxin B by means of productive microorganism Bacillus polymyxa |
-
1984
- 1984-03-24 JP JP59056572A patent/JPH062049B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPS60199398A (en) | 1985-10-08 |
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