JP2005279389A - Method for judging washing period of aqueous system - Google Patents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
Description
本発明は、非酸化性バイオサイドで継続的に処理している水系における、レジオネラ属菌に対する管理方法に関し、具体的には、レジオネラ属菌の水系中での繁殖を効果的、かつ、効率的に抑制することができる水系の洗浄時期の判断方法に関する。 The present invention relates to a management method for Legionella spp. In an aqueous system that is continuously treated with a non-oxidizing biocide, and more specifically, effective and efficient propagation of Legionella spp. The present invention relates to a method for determining a water-based cleaning time that can be suppressed to a low level.
レジオネラ属菌はレジオネラ症の原因微生物として知られており、自然界や人工の水環境に広く生息しているグラム陰性の細菌である。レジオネラ属菌に汚染された冷却塔水や温泉水、循環風呂水などのエアロゾルを吸い込むことによって、レジオネラ属菌に感染し、レジオネラ症を引き起こす場合がある。そのため、「新版 レジオネラ症防止指針」（非特許文献１）が定められ、これらの環境水中のレジオネラ属菌の抑制を行うことが求められている。 Legionella is known as the causative microorganism of Legionella disease, and is a Gram-negative bacterium widely inhabiting the natural world and artificial water environment. Inhalation of aerosols such as cooling tower water, hot spring water, and circulating bath water contaminated with Legionella spp. May infect Legionella spp. And cause Legionellosis. Therefore, the “new edition Legionellosis prevention guideline” (Non-patent Document 1) is established, and it is required to suppress these Legionella species in the environmental water.
このような、水系中のレジオネラ属菌の抑制のために、第四級アンモニウム塩、第四級ホスホニウム化合物等のカチオン系化合物、有機臭素系化合物、イソチアゾリン系化合物、グルタルアルデヒド、ヒドラジン等の非酸化性バイオサイドを継続的に添加する対策が取られる。 In order to suppress legionella in the water system, non-oxidation such as quaternary ammonium salts, cationic compounds such as quaternary phosphonium compounds, organic bromine compounds, isothiazoline compounds, glutaraldehyde, hydrazine, etc. Measures to continuously add sex biocides are taken.
そして、これら非酸化性バイオサイドでの継続的処理により、添加対象の水系中のレジオネラ属菌が実際に抑制されているかどうかについては、その水系の水をサンプルとして、上記「新版 レジオネラ症防止指針」に記載された方法（その一例を図１に示す）等の、公知の培養法によって評価されていた。 Whether or not Legionella spp. In the water system to be added is actually suppressed by the continuous treatment with these non-oxidizing biocides, the above-mentioned “New Legionellosis Prevention Guidelines” And the like (an example of which is shown in FIG. 1).
しかしながら、レジオネラ属菌の培養法による検査では、培養に５〜７日必要であり、水系中にレジオネラ属菌が存在した場合には、その間にその水系中のレジオネラ属菌がさらに増殖して、洗浄が後手に回った結果、何らかの事故等を発生させるおそれがあり、問題となっていた。 However, in the examination by the culture method of Legionella, 5-7 days are required for the culture, and when Legionella is present in the water system, Legionella in the water system further proliferates during that time, As a result of the cleaning being performed later, there was a risk of causing some accidents, which was a problem.
また、培養法では、生きていて、かつ、コロニー形成能を有するレジオネラ属菌のみを検出可能であり、非酸化性バイオサイドで継続的に処理されている水系では、水中に浮遊しているレジオネラ属菌は、通常、非酸化性バイオサイドにより殺菌され、あるいは、コロニー形成能を失うので、培養法では不検出となる。 In the culture method, only Legionella spp. That are alive and have colony-forming ability can be detected. In an aqueous system that is continuously treated with a non-oxidizing biocide, Legionella floating in water is suspended. The genus is usually sterilized by the non-oxidizing biocide or loses the ability to form colonies, so that it is not detected by the culture method.
しかしながら、レジオネラ属菌は、例えば水系内に形成されたバイオフィルム内、あるいは、アメーバ等の原生動物に寄生して、非酸化性バイオサイドから保護された状態で繁殖する性質がある。このようにバイオフィルム内等で繁殖したレジオネラ属菌が水中に漏れ出してきた場合であっても、バイオサイドで継続的に処理されている水系であれば、浮遊している菌はバイオサイドにより殺菌されるため、漏れだしてきた菌が少量の場合には培養法では不検出となる場合が多い。このような場合にはレジオネラ属菌が大繁殖する素地が水系中に形成されているため、対処しないまま放置すると、その後に基準値を遙かに上回る大量の菌が検出されることがあり、問題となる。
本発明は、上記した従来の問題点を改善する、すなわち、非酸化性バイオサイドで継続的に処理している水系において、レジオネラ属菌の水系中での繁殖を効果的、かつ、効率的に抑制することができる水系の洗浄時期の判断方法を提供することを目的とする。 The present invention improves the above-mentioned conventional problems, that is, in an aqueous system continuously treated with a non-oxidizing biocide, the Legionella spp. Can be effectively and efficiently propagated in the aqueous system. It is an object of the present invention to provide a method for determining an aqueous cleaning time that can be suppressed.
ここで、本発明者等は、非酸化性バイオサイドで処理した場合、レジオネラ属菌が死滅してもレジオネラ属菌の遺伝子は比較的長時間に亘り破壊されずに水系に残存していることを見出し、本発明に至った。 Here, when the present inventors treated with non-oxidizing biocide, even if Legionella spp. Die, the gene of Legionella spp. Remains in the water system without being destroyed for a relatively long time. And found the present invention.
すなわち、本発明は、上記課題を解決するために、非酸化性バイオサイドで継続的に処理している水系において、レジオネラ属菌の検査を、遺伝子検査法を用いて定期的に行い、その結果を基に水系の洗浄時期を判断する水系の洗浄時期の判断方法である。 That is, the present invention, in order to solve the above problems, in the water system that is continuously treated with a non-oxidizing biocide, Legionella spp. Is periodically tested using a genetic test method, and as a result, This is a method for determining the cleaning time of an aqueous system based on the above.
ここで、高濃度の非酸化性バイオサイドで洗浄後、低濃度の非酸化性バイオサイドで継続的に処理している水系において、水系を洗浄処理する前後における水系の状態（推定される状態）とその状態における遺伝子検査法及び培養法によるレジオネラ属菌の検査結果を表１に示す。 Here, after washing with a high concentration of non-oxidizing biocide, in an aqueous system that is continuously treated with a low concentration of non-oxidizing biocide, the state of the aqueous system before and after washing the aqueous system (estimated state) Table 1 shows the test results of Legionella spp. By genetic testing and culture methods in that state.
表１中、状態１では水系全体がレジオネラ属菌で汚染されており、培養法と遺伝子検査法との両者でレジオネラ属菌が陽性となる。そこで、非酸化性バイオサイドを用いて水系の洗浄を行うと、系内のレジオネラ属菌は死滅するが、その死骸中の遺伝子は水中に残存した状態となり、培養法では陰性、遺伝子検査法では陽性という状態となる（状態２）。 In Table 1, in state 1, the entire water system is contaminated with Legionella, and Legionella is positive in both the culture method and the genetic test method. Therefore, when the aqueous system is washed with non-oxidizing biocide, Legionella spp. In the system are killed, but the genes in the dead body remain in water, negative in the culture method, negative in the genetic test method It becomes a state of being positive (state 2).
この状態でしばらく水系の運転を続けると、死骸中の遺伝子の分解、水の入れ替わり等によりレジオネラ属菌遺伝子は水系内からなくなり、遺伝子検査の結果も陰性となる（状態３） If the water system is operated for a while in this state, the gene of Legionella will disappear from the water system due to the degradation of the gene in the carcass, the replacement of water, etc., and the result of the genetic test will be negative (State 3)
その後、系内が徐々に汚れ、バイオフィルムが形成されるようになると、バイオフィルム内でレジオネラ属菌が増殖し、その一部が浮遊菌として水中に漏れ出すようになる（状態４）。この状態では、水中に漏れ出した浮遊レジオネラ属菌は、バイオサイドの殺菌効果で死滅するため、培養法では陰性となるが、レジオネラ属菌の遺伝子は短時間では分解しないので、遺伝子検査では検出されて陽性となる。この水系をさらにそのまま放置すると、バイオフィルム内のレジオネラ属菌の増殖が盛んになり、バイオサイドの殺菌効果がバイオフィルムから供給されるレジオネラ属菌の量に追いつかなくなるので、培養法でもレジオネラ属菌が陽性となる（状態５）。 Thereafter, when the system is gradually soiled and a biofilm is formed, Legionella spp. Grow in the biofilm, and a part of it leaks into the water as floating bacteria (state 4). In this state, floating Legionella spp. That have leaked into the water are killed by the biocidal bactericidal effect, making it negative in the culture method, but Legionella spp. And become positive. If this water system is further left as it is, the growth of Legionella in the biofilm will flourish and the bactericidal effect of the biocide will not be able to catch up with the amount of Legionella supplied from the biofilm. Becomes positive (state 5).
状態５となるまで放置すると、水系内のレジオネラ属菌の汚染はかなり進んだ状態となり、水系の洗浄に際し、高濃度の薬品を長時間接触させる必要が生じる等の手間が掛かるばかりか、次回の洗浄までの期間、生きたレジオネラ属菌が水中に浮遊して存在することとなり、エアロゾル等の飛散によりレジオネラ属菌に感染する危険性が高くなる。 If left to state 5, the contamination of Legionella spp. In the water system will be considerably advanced, and not only will it take time to contact the chemical with high concentration for a long time when cleaning the water system, During the period until washing, living Legionella bacteria float and exist in water, and there is a high risk of infection with Legionella bacteria due to scattering of aerosols and the like.
本発明は、生きたレジオネラ属菌が浮遊して水中に存在するようになる前の、上記表１の状態４となった時期を検知する方法を提供するものであり、その時期に水系を洗浄することで、生きたレジオネラ属菌がエアロゾル等により環境中に飛散することを避けることができ、さらに、上記状態５となる前に洗浄を行うことが可能となるので、従来の培養法による検知後の洗浄よりも簡易な処理でも充分な効果を得ることが可能となる。 The present invention provides a method for detecting the time when the live Legionella spp. Floats and becomes in the water before the state 4 in Table 1 is reached, and the water system is washed at that time. By doing so, it is possible to avoid that living Legionella spp. Are scattered in the environment by aerosols and the like, and further, washing can be performed before the state 5 is reached. A sufficient effect can be obtained even by a simpler treatment than the subsequent cleaning.
本発明によれば、遺伝子検査法の結果を用いて洗浄の要否の判断を行うため、従来の培養法（結果が出るまで５〜７日、あるいは１０日必要）では検出できなかった、水系中に存在しているレジオネラ属菌の死菌由来の遺伝子であっても検出できるので、水系中の、例えばバイオフィルム内などで、局所的にレジオネラ属菌が繁殖しているが、水中に浮遊している菌は生菌としては存在していない場合であっても、水系中のレジオネラ属菌の生存を検出することが可能となり、それら局所的に存在するレジオネラ属菌が水系全体に広がる前にその存在を知ることができ、さらに、遺伝子検査法ならではの迅速な検査（２〜１０時間程度で検出）が可能であることも相俟って、水系の洗浄が後手に回ることがなく、水系のレジオネラ属菌の安心で、安定した管理が可能となり、レジオネラ感染症の発生などと云う大事故を未然に防止することができる。 According to the present invention, since the necessity of washing is determined using the result of the genetic test method, an aqueous system that could not be detected by the conventional culture method (5 to 7 days or 10 days are required until the result is obtained). Legionella spp. Can be detected even if it is a gene derived from killed Legionella spp. Present in the water system, for example in a biofilm, but Legionella spp. Even if the bacterium is not present as a living bacterium, it is possible to detect the survival of Legionella in the water system, before the locally existing Legionella genus spreads throughout the water system. In addition to being able to know its presence, and also being able to perform rapid testing (detection in about 2 to 10 hours) unique to genetic testing, washing of the water system does not go to the back, Relief of Legionella spp. , It is possible to stable management, a large accident referred to as the occurrence of Legionella infection can be prevented in advance.
本発明において、対象となる水系は非酸化性バイオサイドで継続的に処理されている水系であることが必要である。ここで、非酸化性バイオサイドで継続的に処理されているとは、水系中の非酸化性バイオサイドの濃度が一定値以上に保たれている、あるいは、非酸化性バイオサイドの水系への添加が１０日に１回より高い頻度で定期的に行われていることを指す。なお、後者のバイオサイドの添加は、必ずしも厳密に規定する必要はなく、例えばレジオネラ属菌が繁殖しやすい夏期での添加間隔を、冬季での添加間隔より短くするなど、水系の条件に応じて変動させて良く、この場合にも本発明に含まれる。 In the present invention, the target aqueous system needs to be an aqueous system that is continuously treated with a non-oxidizing biocide. Here, the continuous treatment with the non-oxidizing biocide means that the concentration of the non-oxidizing biocide in the aqueous system is maintained at a certain value or more, or the non-oxidizing biocide into the aqueous system It means that the addition is regularly performed more frequently than once every 10 days. The latter biocide addition does not necessarily have to be strictly defined. For example, the addition interval in summer when Legionella spp. Is easy to reproduce, depending on the conditions of the water system, such as making it shorter than the addition interval in winter. It may be varied, and this case is also included in the present invention.
本発明において用いられる非酸化性バイオサイドとしては、第四級アンモニウム塩、第四級ホスホニウム化合物等のカチオン系化合物、有機臭素系化合物、イソチアゾリン系化合物、グルタルアルデヒド、ヒドラジンなどの非酸化性薬品であって、レジオネラ属菌に対して有効なバイオサイドであればよく、これらを単独で、あるいは複数を組み合わせて用いても良い。 Non-oxidizing biocides used in the present invention include cationic compounds such as quaternary ammonium salts and quaternary phosphonium compounds, organic bromine compounds, isothiazoline compounds, glutaraldehyde, hydrazine and other non-oxidizing chemicals. Any biocide that is effective against Legionella bacteria may be used, and these may be used alone or in combination.
なお、本発明における非酸化性バイオサイドでの継続的処理には、水中で次亜塩素酸及び／または次亜臭素酸を放出する化合物、二酸化塩素、過酸化物、オゾン等の酸化性バイオサイドは併用することができない。このような酸化性バイオサイドを併用すると、水系中のレジオネラ属菌の死菌由来の遺伝子や、培養法では検出されない、コロニー形成能を失ったレジオネラ属菌由来の遺伝子が、これら酸化性バイオサイドによって酸化、分解されてしまうので、上記のように水系内の特定の箇所でレジオネラ属菌が繁殖している場合等での検知ができなくなってしまう。 In addition, the continuous treatment with the non-oxidizing biocide in the present invention includes an oxidizing biocide such as a compound that releases hypochlorous acid and / or hypobromite in water, chlorine dioxide, peroxide, ozone, etc. Cannot be used together. When such oxidative biocide is used in combination, genes derived from killed Legionella spp. In the aquatic system and genes derived from Legionella spp. As a result, it becomes impossible to detect when Legionella spp. Are propagated at a specific location in the water system as described above.
本発明ではレジオネラ属菌の検査を、遺伝子検査法を用いて行うことが、死菌の存在や培養法では検出されない、コロニー形成能を失ったレジオネラ属菌の存在を検出するために、必要である。 In the present invention, the detection of Legionella genus is performed using a genetic test method in order to detect the presence of dead bacteria and the presence of Legionella genus that has lost colony forming ability, which is not detected by the culture method. is there.
本発明における遺伝子検査法とは、検査対象の細菌に応用できる遺伝子検査法から選択すればよい。例えば、ＰＣＲ法（ポリメラーゼ連鎖反応法）、ＬＡＭＰ法、あるいはＰＡＬＳＡＲ法として知られる核酸の増幅・検知法を用いた遺伝子検査法が挙げられ、それぞれ例えば特公平４−６７９６０号公報、再公表特許ＷＯ００／２８０８２、あるいは、特開２０００−２０１６８７公報に記載されていることが知られている。これらはいずれも２時間〜１０時間程度で、検査対象の細菌の遺伝子の特徴部分を多量に複製または検知することができる方法で、その後、電気泳動等の手段により、検査対象の細菌の遺伝子の有無を容易かつ迅速に判定することができる。 What is necessary is just to select the genetic test method in this invention from the genetic test methods applicable to the test | inspection bacterium. For example, genetic testing methods using nucleic acid amplification / detection methods known as PCR method (polymerase chain reaction method), LAMP method, or PALSAR method can be mentioned, for example, Japanese Patent Publication No. 4-67960 and republished patent WO 00 / 28082 or JP-A 2000-201687 is known. These are methods that can replicate or detect a large amount of the characteristic part of the bacterial gene to be examined in about 2 hours to 10 hours, and then, by means of electrophoresis or the like, Presence / absence can be determined easily and quickly.
本発明における水系の洗浄とは、水系内のレジオネラ属菌を確実に駆除できる条件で、バイオサイドを用いた化学洗浄処理を指す。このとき、物理的洗浄やろ過、浮遊物除去、水の一部あるいは全部交換等を併用しても良く、これにより、水系の洗浄の効果をより高いものとすることができる。 The aqueous cleaning in the present invention refers to a chemical cleaning treatment using biocide under conditions that can surely eliminate Legionella bacteria in the aqueous system. At this time, physical washing, filtration, removal of suspended solids, replacement of part or all of water, and the like may be used in combination, whereby the effect of washing with water can be further enhanced.
本発明において、水系の洗浄に用いるバイオサイドは、継続的処理で用いているバイオサイドを用いても良く、また、他の非酸化性バイオサイドや酸化性バイオサイドを用いても良く、また、互いに影響して効果が減じない限りにおいてこれらを併用しても良い。酸化性バイオサイドを用いるとレジオネラ属菌駆除後に水系内に残留するレジオネラ属菌の死菌由来の遺伝子が遺伝子検査法で検出されないように分解されるので、水系の洗浄以降の誤検出を防止することができるので好ましい。 In the present invention, the biocide used for aqueous cleaning may be the biocide used in the continuous treatment, or may use other non-oxidative biocide or oxidative biocide, These may be used in combination as long as the effects are not reduced by mutual influence. Use of an oxidizing biocide will prevent the detection of Legionella spp. Dead genes remaining in the water system after extermination of Legionella so that it will not be detected by genetic testing, thus preventing false detection after washing the water system This is preferable.
この水系の洗浄でのバイオサイドの添加は水系内のレジオネラ属菌を充分に除去できるような添加濃度となるように行う必要がある。従って、継続的処理で用いているバイオサイドを水系の洗浄でも用いる場合には、継続的処理での水系内濃度よりも高い濃度となるようにして添加する。 It is necessary to add the biocide in this water-based washing so that the added concentration can sufficiently remove Legionella spp. In the water system. Therefore, when the biocide used in the continuous treatment is also used in the washing of the aqueous system, it is added so as to have a concentration higher than the concentration in the aqueous system in the continuous treatment.
本発明において上記の水系の洗浄を行う、水系の洗浄時期を判断するには、定期的に行う遺伝子検査法を用いて行う。具体的には水系から採取した水をサンプルとして、そのサンプル中に遺伝子検査法によってレジオネラ属菌の遺伝子が検出されていなかった状態から検出されるようになった場合（陰性から陽性に転じた場合）に、その水系中のどこかにレジオネラ属菌が生存しているとして、水系の洗浄を行うこととする。 In the present invention, in order to determine the water-based cleaning time for performing the above-described water-based cleaning, a periodic genetic test method is used. Specifically, water collected from the water system is used as a sample, and when the gene from the Legionella genus has not been detected by genetic testing in the sample (when it turns from negative to positive) ), It is assumed that Legionella bacteria are alive somewhere in the water system, and the water system is washed.
上記遺伝子検査法において「定期的」とは、具体的は、１０〜６０日に１回程度遺伝子検査法を行うことであって、その間隔は必ずしも厳密に規定する必要はなく、例えばレジオネラ属菌が繁殖しやすい夏期での間隔を、冬季での間隔より短くするなど、水系の条件に応じて変動させて良く、この場合にも本発明に含まれる。 In the above genetic testing method, “periodic” specifically means that the genetic testing method is performed about once every 10 to 60 days, and the interval does not necessarily have to be strictly defined. For example, Legionella spp. It is possible to change the interval in the summer season when the fish is easy to breed, depending on the conditions of the water system, such as to make it shorter than the interval in the winter season, and this case is also included in the present invention.
なお、遺伝子検査法に加えて、培養法による検査を平行して行っても良く、遺伝子検査法の結果が陽性に転じた際に、培養法では陰性であることが確認できれば、水系内が表１で状態４として示される汚れ具合であることが確定できるため、このとき、簡易な洗浄方法をとっても充分な洗浄効果を得ることができる。 In addition to the genetic test method, the culture method test may be performed in parallel. If the result of the genetic test method turns positive, it can be confirmed that the culture system is negative. 1 can be determined to be the state of contamination shown as state 4, and at this time, a sufficient cleaning effect can be obtained even with a simple cleaning method.
＜基礎検討＞
基礎検討として、非酸化性バイオサイドによる洗浄によっても水中のレジオネラ属菌の遺伝子が容易には分解されないことを下記のようにして確認した。
<Basic study>
As a basic study, it was confirmed as follows that the gene of Legionella spp. In water was not easily degraded by washing with non-oxidizing biocide.
非酸化性のバイオサイドとして、ヒドラジン（６０％水加ヒドラジン溶液、大塚化学社製）、グルタルアルデヒド（５０％水溶液、キシダ化学社製。「ＧＡ」とも云う）、５−クロロ−２−メチル−４−イソチアゾリン−３−オン（ゾーネンＣ、ケミクレア社製。「ＣＭＩ」とも云う）、塩化ベンザルコニウム（５０％塩化ベンザルコニウム溶液、キシダ化学社製。「ＢＫＣ」とも云う）および２−ブロモ−２−ニトロプロパン−１、３−ジオ−ル（ＮＫ−１３４、ケイ・アイ化成社製。「ＢＮＰＤ」とも云う）を、また、比較のため、酸化性バイオサイドとして過酸化水素（３１％水溶液、三菱瓦斯化学社製）、及び、次亜塩素酸ナトリウムを用いて試験を行った。 As non-oxidizing biocides, hydrazine (60% hydrazine solution, manufactured by Otsuka Chemical Co., Ltd.), glutaraldehyde (50% aqueous solution, manufactured by Kishida Chemical Co., Ltd., also referred to as “GA”), 5-chloro-2-methyl- 4-isothiazolin-3-one (Zonen C, manufactured by Chemicrea Inc., also referred to as “CMI”), benzalkonium chloride (50% benzalkonium chloride solution, manufactured by Kishida Chemical Co., Ltd., also referred to as “BKC”) and 2-bromo 2-Nitropropane-1,3-diol (NK-134, manufactured by KAI Kasei Co., Ltd., also referred to as “BNPD”), and for comparison, hydrogen peroxide (31%) as an oxidizing biocide. The test was performed using an aqueous solution, manufactured by Mitsubishi Gas Chemical Co., Ltd.) and sodium hypochlorite.
一晩汲み置きした水道水をそれぞれ１Ｌずつ（ただし、過酸化水素の試験を行うものは９５０ｍＬ）容れた容量が１Ｌのポリプロピレン製容器に、レジオネラ属菌（Ｌｅｇｉｏｎｅｌｌａ ｐｎｅｕｍｏｐｈｉｌａ ｓｅｒｏｇｒｏｕｐ １ Ｐｈｉｌａｄｅｌｐｈｉａ−１（ＡＴＣＣ３３１５２）株）をおよそ１０7ＣＦＵ／１００ｍＬとなるようにそれぞれ懸濁させ試験水とした。 Each 1L of tap water drawn overnight (950mL for hydrogen peroxide test) is placed in a 1L polypropylene container with Legionella pneumophila serogroup 1 Philadelphia-1 (ATCC 33152) The test water was suspended at about 10 7 CFU / 100 mL.
これらそれぞれに、６０重量％水加ヒドラジン溶液を５０ｍｇ（このときヒドラジン濃度は１９ｍｇ／Ｌとなる）、５０重量％グルタルアルデヒド水溶液を４０ｍｇ（このときのグルタルアルデヒド濃度は２０ｍｇ／Ｌ）、５０重量％塩化ベンザルコニウム溶液を４０ｍｇ（このときの塩化ベンザルコニウム濃度は２０ｍｇ／Ｌ）、上記ゾーネンＣを２０ｍｇ（５−クロロ−２−メチル−４−イソチアゾリン−３−オン濃度は２ｍｇ／Ｌとなる）、ＮＫ−１３４を１０ｍｇ（このときの２−ブロモ−２−ニトロプロパン−１、３−ジオ−ル濃度は１０ｍｇ／Ｌ）、３１％過酸化水素水を５０ｇ（過酸化水素濃度は１５５００ｍｇ／Ｌ）をそれぞれに添加した。また、次亜塩素酸ナトリウム溶液（１２％、キシダ化学）を添加した系では、試験水の残留塩素濃度が０．２ｍｇ／Ｌを維持するように調整した。 For each of these, 50 mg of a 60 wt% hydrazine solution (the hydrazine concentration is 19 mg / L at this time), 40 mg of a 50 wt% aqueous solution of glutaraldehyde (the glutaraldehyde concentration at this time is 20 mg / L), 50 wt% 40 mg of benzalkonium chloride solution (concentration of benzalkonium chloride at this time is 20 mg / L) and 20 mg of zonen C (concentration of 5-chloro-2-methyl-4-isothiazolin-3-one is 2 mg / L) ), 10 mg of NK-134 (2-bromo-2-nitropropane-1,3-diol concentration at this time is 10 mg / L), 50 g of 31% hydrogen peroxide (hydrogen peroxide concentration is 15500 mg / L). L) was added to each. Further, in a system to which a sodium hypochlorite solution (12%, Kishida Chemical) was added, the residual chlorine concentration of the test water was adjusted to maintain 0.2 mg / L.
これらを室温で放置し、２４時間後および４８時間後に採水容器に５００ｍＬずつ採水し、これらのレジオネラ属菌の生菌数についてＰＣＲ法による遺伝子検査法及び培養法により検査を行った。その結果を表２および表３に示す。 These were allowed to stand at room temperature, and after 24 hours and 48 hours, 500 mL each was collected in a water collection container, and the viable count of these Legionella spp. Was examined by a genetic test method and a culture method using the PCR method. The results are shown in Tables 2 and 3.
上記のように、非酸化性バイオサイド、酸化性バイオサイドを問わず、処理開始後２４時間後にはいずれの系も培養ではレジオネラ属菌は陰性となり、生菌がいないことが確認された。一方、遺伝子検査法では、非酸化性バイオサイドで処理した系ではすべて処理２４時間後、あるいは４８時間後であってもレジオネラ属菌の遺伝子が検出され、また、酸化性バイオサイドで処理した系ではレジオネラ属菌の遺伝子が検出されないことが判った。 As described above, regardless of whether non-oxidizing biocide or oxidizing biocide, 24 hours after the start of treatment, it was confirmed that Legionella sp. On the other hand, in the gene testing method, Legionella gene is detected in all the systems treated with non-oxidizing biocide even after 24 hours or 48 hours, and the system treated with oxidizing biocide. It was found that the gene of Legionella was not detected.
これら結果より、非酸化性バイオサイドで処理した場合、レジオネラ属菌が死滅してもレジオネラ属菌の遺伝子は比較的長時間に亘り破壊されずに水系に残存し、その結果、遺伝子検査法で検出可能であることが判る。また、これに対して酸化性バイオサイドを用いた場合には、レジオネラ属菌の死菌由来の遺伝子は速やかに酸化・分解されてしまうので、遺伝子検査法では検出されなくなることが判る。 From these results, when treated with a non-oxidizing biocide, the Legionella gene remains in the water system without being destroyed for a relatively long time even if the Legionella genus is killed. It turns out that it can detect. In contrast, when the oxidizing biocide is used, it can be seen that the gene derived from the killed Legionella genus is rapidly oxidized and decomposed, so that it cannot be detected by the genetic test method.
なお、表２及び表３中、ＰＣＲ法による検出は次のように行った（後述する実際の水系での検討でも同様に実施）。 In Tables 2 and 3, the detection by the PCR method was performed as follows (the same applies to the examination in an actual aqueous system described later).
すなわち、試料水各５００ｍＬをメンブレンフィルター（孔径：０．４５μｍ）を用いてろ過したのち続けて、５０ｍＬの滅菌脱イオン水をろ過してメンブレンフィルターを濯いだ。 That is, each 500 mL of sample water was filtered using a membrane filter (pore size: 0.45 μm), and then 50 mL of sterilized deionized water was filtered to rinse the membrane filter.
ろ過捕捉物の付着した上記メンブレンフィルターを、滅菌済みの５０ｍＬ容のスクリューキャップ付き遠心沈殿管に入れ、滅菌脱イオン水を５ｍｌ加えてボルテックスタイプミキサーで５分間攪拌して捕捉物を再懸濁させた。 Place the membrane filter with the filtered trapped substance in a sterilized 50 mL centrifuge tube with screw cap, add 5 ml of sterilized deionized water, and stir for 5 minutes with a vortex mixer to resuspend the captured substance. It was.
次いで、上記で得た再懸濁液２ｍＬを２ｍＬ容のマイクロチューブに移し、４℃、１４０００×ｇで１０分間遠心処理を行った。 Next, 2 mL of the resuspension obtained above was transferred to a 2 mL microtube, and centrifuged at 14,000 × g for 10 minutes at 4 ° C.
その上清１９６０μＬを取り除き、５０ｍＭ水酸化ナトリウム水溶液を５０μＬ加え、沸騰水中（１００℃）にこのマイクロチューブを浸漬し、１５分間加熱して遺伝子を菌体外に抽出した。次いで、マイクロチューブを氷冷した後、１Ｍ−Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．０）を８μＬ添加して中和し、４℃の温度下で、１４０００×ｇで１０分間遠心処理し、その上清をＰＣＲ用の試料として用いた。 1960 μL of the supernatant was removed, 50 μL of 50 mM sodium hydroxide aqueous solution was added, the microtube was immersed in boiling water (100 ° C.), and heated for 15 minutes to extract the gene outside the cells. Next, the microtube was ice-cooled, neutralized by adding 8 μL of 1M-Tris-HCl buffer (pH 7.0), centrifuged at 14000 × g for 10 minutes at a temperature of 4 ° C. Kiyoshi was used as a sample for PCR.
ＰＣＲに用いるプライマーはＹａｍａｍｏｔｏら（「環境水中のレジオネラ属菌の検出方法としての、培養法、蛍光抗体染色法、ＰＣＲ法の比較」(Yamamoto, H., Y. Hashimoto and T. Ezaki (1993) Comparison of detection methods for Legionella species in environmental water by colony isolation, fluorescent antibody staining, and polymerase chain reaction. Microbiol. Immunol., 37, 617-622)が報告している、レジオネラ属細菌の１６Ｓ ｒＲＮＡをターゲットとするＬＥＧ４４８−Ａ（５’−ＧＡＧ ＧＧＴ ＴＧＡ ＴＡＧ ＧＴＴ ＡＡＧ ＡＧＣ−３’）（配列番号１）およびＬＥＧ８５４−Ｂ（５’−ＣＧＧ ＴＣＡ ＡＣＴ ＴＡＴ ＣＧＣ ＧＴＴ ＴＧＣ Ｔ−３’）（配列番号２）を用いた。 Primers used for PCR were Yamamoto et al. (“Comparison of culture method, fluorescent antibody staining method, PCR method as a method for detecting Legionella spp. In environmental water” (Yamamoto, H., Y. Hashimoto and T. Ezaki (1993) Comparison of detection methods for Legionella species in environmental water by colony isolation, fluorescent antibody staining, and polymerase chain reaction. Microbiol. Immunol., 37, 617-622), targeting 16S rRNA of Legionella bacteria For LEG448-A (5′-GAG GGT TGA TAG GTT AAG AGC-3 ′) (SEQ ID NO: 1) and LEG854-B (5′-CGG TCA ACT TAT CGC GTT TGC T-3 ′) (SEQ ID NO: 2) It was.
ＰＣＲの反応液はＴａＫａＲａ Ｅｘ Ｔａｑ Ｈｏｔ Ｓｔａｒｔ Ｖｅｒｓｉｏｎ（タカラバイオ社製）を用いて調整した。反応液の組成は１４．３７５μＬの滅菌蒸留水に２．５μＬの１０× Ｅｘ Ｔａｑ緩衝液（ｂｕｆｆｅｒ）、２μＬのｄ−ＮＴＰ ｍｉｘ、０．５μＬのＬＥＧ４４８−Ａプライマー溶液（１０μＭ）、０．５μＬのＬＥＧ８５４−Ｂプライマー溶液（１０μＭ）および０．１２５μＬのＥｘ Ｔａｑ ｐｏｌｙｍｅｒａｓｅを混合し、最後に５μＬの核酸抽出液を添加して全量を２５μＬとした。 The PCR reaction solution was prepared using TaKaRa Ex Taq Hot Start Version (manufactured by Takara Bio Inc.). The composition of the reaction solution was 2.5 μL of 10 × Ex Taq buffer (buffer), 2 μL of d-NTP mix, 0.5 μL of LEG448-A primer solution (10 μM), 0.5 μL in 14.375 μL of sterile distilled water. LEG854-B primer solution (10 μM) and 0.125 μL of Ex Taq polymerase were added, and finally 5 μL of nucleic acid extract was added to make a total volume of 25 μL.
サーマルサイクラーの設定は、最初の熱変性として９４℃で９０秒、続けて９４℃で３０秒、６５℃で６０秒及び７２℃で６０秒の操作を１サイクルとして５サイクル行った。その後、９４℃で３０秒、６５℃で３０秒及び７２℃で６０秒の操作を１サイクルとして３５サイクル行い、最後に７２℃で４分の反応を行った。 The thermal cycler was set for 5 cycles, with the initial heat denaturation at 94 ° C. for 90 seconds, followed by 94 ° C. for 30 seconds, 65 ° C. for 60 seconds, and 72 ° C. for 60 seconds. Thereafter, 35 cycles were carried out, with one cycle consisting of 94 ° C. for 30 seconds, 65 ° C. for 30 seconds and 72 ° C. for 60 seconds, and finally a reaction was carried out at 72 ° C. for 4 minutes.
増幅産物の検出はアガロースゲル（１．５重量％）電気泳動で行った。泳動後のゲルをＳＹＢＲ Ｇｒｅｅｎ Ｉ（ＢＭＰ）を用いて染色し、２５４ｎｍのトランスイルミネーター上で、レジオネラ属菌由来である４３０ｂｐの増幅産物の有無を観察し、増幅産物が観察された場合を「陽性」と判断した。 The amplification product was detected by agarose gel (1.5% by weight) electrophoresis. The gel after electrophoresis was stained with SYBR Green I (BMP), and the presence or absence of a 430 bp amplification product derived from Legionella was observed on a 254 nm transilluminator. Positive.
また、表２及び表３における培養法による検査は次のようにして行ったものである（後述する実際の水系での検討でも同様に実施）。 Moreover, the test | inspection by the culture method in Table 2 and Table 3 was performed as follows (it is implemented similarly also in the examination in the actual water system mentioned later).
上記遺伝子検査法と同様にしてメンブレンフィルターによるろ過濃縮を行った後の再懸濁液１ｍＬを用いて、「新版 レジオネラ症防止指針」に記載された冷却遠心濃縮法を用いた平板培養法のフローチャート（図１）に沿って定めた、図２に示すフローチャートに従ってＧＶＰＣα培地を用いて行った。 Flowchart of the plate culture method using the cooling centrifugal concentration method described in “New Edition Legionellosis Prevention Guideline” using 1 mL of the resuspension after filtration concentration with a membrane filter in the same manner as the genetic test method above. This was carried out using GVPCα medium according to the flowchart shown in FIG. 2 determined along (FIG. 1).
＜実際の水系での検討＞
レジオネラ属菌が繁殖した、保有水量：１０ｍ3、冷凍能力：２００ＲＴの循環冷却水系に２００ｍｇ／Ｌのグルタルアルデヒドを投入して水系内を除菌洗浄した後、５−クロロ−２−メチル−４−イソチアゾリン−３−オンを水系内の濃度が計算上２ｍｇ／Ｌを維持するように補給水に対して連続的に供給する処理を行った。なお、水系の洗浄後の全換水は行わず、冷却水系の濃縮倍率が約５倍となるように濃縮管理を行った。
<Examination in actual water system>
Legionella spp. Propagated, retained water volume: 10 m 3 , freezing capacity: 200 mg / L glutaraldehyde was added to a circulating cooling water system of 200 RT to disinfect the water system, and then 5-chloro-2-methyl-4 -The treatment which supplies isothiazolin-3-one continuously with respect to makeup water so that the density | concentration in a water system may maintain 2 mg / L in calculation is performed. In addition, concentration management was performed so that the total water exchange after washing the water system was not performed, and the concentration ratio of the cooling water system was about 5 times.
この水系の管理開始初日にＰＣＲ法及び培養法によってレジオネラ属菌の検査を行うために水系の水をサンプルとして採取した（以下、同様に同じ箇所でサンプル採取を行った）後、５０重量％−グルタルアルデヒド水溶液を４ｋｇ投入することにより水系の洗浄（以下、同じ条件）を行った。 In order to test Legionella by the PCR method and the culture method on the first day of the management of this aqueous system, aqueous water was sampled (hereinafter, sampled at the same location in the same manner), and then 50% by weight− Aqueous washing (hereinafter, the same conditions) was performed by adding 4 kg of glutaraldehyde aqueous solution.
水系の洗浄を行った次の日（２日目）にも、サンプルを採取し、ＰＣＲ法及び培養法によってレジオネラ属菌の検査を行った。その結果ＰＣＲ法ではレジオネラ属菌の遺伝子の存在が検出されたが、培養法ではレジオネラ属菌は検出されなかった（表４参照）ため、水系中に残存するレジオネラ属菌の遺伝子は死菌由来の遺伝子であると判断した（このことは７日目に採取したサンプルでは、ＰＣＲ法及び培養法の両者で陰性となったことで確認された）。 On the next day (second day) after washing the aqueous system, a sample was collected and examined for Legionella by the PCR method and the culture method. As a result, the presence of Legionella gene was detected by the PCR method, but Legionella gene was not detected by the culture method (see Table 4). Therefore, the Legionella gene remaining in the water system was derived from dead bacteria. (This was confirmed by the fact that the sample collected on the 7th day was negative in both the PCR method and the culture method).
また、その後、４箇月後まで、１箇月ごとに定期的にサンプルを採取し、ＰＣＲ法及び培養法によってレジオネラ属菌の検査を行ったが、両者ともに陰性であった。 In addition, after that, until 4 months later, samples were taken periodically every month and tested for Legionella by PCR and culture methods. Both were negative.
管理開始５箇月後にはＰＣＲ法では検出されたものの、培養法では陰性となったため、そのまま放置したところ、６箇月後には、生菌数が１．６×１０3個／１００ｍＬと云う大量のレジオネラ属菌が存在する状況（培養法で）となっていることが判り、２回目の水系の洗浄を行った（以上、従来技術例）。 Although it was detected by the PCR method 5 months after the start of the control but became negative by the culture method, when left as it was, a large amount of Legionella whose viable count was 1.6 × 10 3 cells / 100 mL after 6 months. It was found that the genus bacteria existed (by the culture method), and the second aqueous cleaning was performed (the conventional example).
この２回目の洗浄後から、本発明の水系の洗浄時期の判断方法に従って、定期的な遺伝子検査法の結果を基に水系の洗浄時期を判断した。具体的には遺伝子検査法を用いて水系中のレジオネラ属菌の遺伝子が検出されたときに水系の洗浄を行うこととした。 After this second washing, the washing time of the water system was determined based on the results of the periodic genetic test method according to the method for judging the washing time of the water system of the present invention. Specifically, the water system was washed when the gene of Legionella in the water system was detected using the genetic test method.
その結果、２回目の水系の洗浄後は、培養法ではレジオネラ属菌は一度も検出されず、本発明によれば、水系内のレジオネラ属菌を効果的に抑制できることが確認された。 As a result, after the second washing of the aqueous system, Legionella was never detected by the culture method, and according to the present invention, it was confirmed that Legionella in the aqueous system can be effectively suppressed.
本発明は、水系のレジオネラ属菌を、後手に回ることがなく確実に抑制することができるので、レジオネラ属菌の抑制が求められる水系に、広く応用することができる。 INDUSTRIAL APPLICATION Since this invention can suppress reliably an aqueous | water-based Legionella genus, without turning back, it can apply widely to the aqueous system by which suppression of Legionella genus bacteria is calculated | required.
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