JP5885440B2 - Concomitant drug sensitivity test - Google Patents
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Description
本発明は、抗菌薬を併用投与する際の菌に対する薬剤併用効果の適否を判定する、薬剤感受性検査法に関する。 The present invention relates to a drug sensitivity test method for determining the suitability of a drug combination effect against bacteria when an antibacterial drug is administered in combination.
感染症における化学療法は、原則として抗菌薬を単剤で用いる治療が行われている。そのため、治療に用いる抗菌薬を選定するために、その候補となる抗菌薬の単剤での抗菌活性を薬剤感受性検査法で測定し、抗菌薬の効果の適否を判定している。その試験方法としては、微量液体希釈法、寒天平板希釈法、ディスク拡散法などが知られている。 As a general rule, chemotherapy for infectious diseases is performed using an antibacterial agent alone. Therefore, in order to select an antibacterial agent to be used for treatment, the antibacterial activity of a single candidate antibacterial agent is measured by a drug sensitivity test method to determine whether the antibacterial agent is appropriate. As the test method, a micro liquid dilution method, an agar plate dilution method, a disk diffusion method and the like are known.
この試験結果の評価として、抗菌薬の治療対象となる微生物に対した発育を阻止する最小濃度（ＭＩＣ：ｍｉｎｉｍｕｍ ｉｎｈｉｂｉｔｏｒｙ ｃｏｎｃｅｎｔｒａｔｉｏｎ）を基に、投与した時にその抗菌薬が有効に作用するか否かを判断する基準の世界的なガイドラインが、ＣＬＳＩ（Ｃｌｉｎｉｃａｌ ａｎｄ Ｌａｂｏｒａｔｏｒｙ Ｓｔａｎｄａｒｄｓ Ｉｎｓｔｉｔｕｔｅ）から公表されている（非特許文献１）。 As an evaluation of this test result, whether or not the antibacterial drug acts effectively when administered based on the minimum concentration (MIC) that inhibits the growth of the antimicrobial drug against the microorganisms to be treated. A global guideline for determining the criteria is published by CLSI (Clinical and Laboratory Standards Institute) (Non-patent Document 1).
この判断基準は、感性（Ｓ：治療効果が期待できる）、中間（Ｉ：投与量により治療効果が期待できる、効果の有無の緩衝域）、耐性（Ｒ：治療効果が期待できない）の３種類に区分され、感染症の起因菌に対して抗菌薬のＭＩＣが感性の値であれば投与することで治療効果が期待でき、耐性の値であれば投与しても治療効果は見込まれず、該起因菌は該抗菌薬に対して耐性菌と呼ばれる。 There are three types of judgment criteria: sensitivity (S: a therapeutic effect can be expected), intermediate (I: a therapeutic effect can be expected depending on the dosage, buffer range of presence or absence of effect), and tolerance (R: a therapeutic effect cannot be expected). If the MIC of the antibacterial agent is sensitive to the infectious disease-causing bacteria, it can be expected to have a therapeutic effect, and if it is resistant, no therapeutic effect is expected even if administered. The causative bacteria are called resistant bacteria to the antibacterial drug.
一方、日本国内においては、抗菌薬の国内での常用投与量を基準にして、呼吸器感染症および敗血症（非特許文献２）ならびに尿路感染症（非特許文献３）を対象とした抗菌薬のブレイクポイントを算定するガイドラインが、日本化学療法学会抗菌薬感受性測定法検討委員会から報告されている。 On the other hand, in Japan, antibacterial drugs targeting respiratory infections and sepsis (Non-patent Document 2) and urinary tract infections (Non-patent Document 3) based on the domestic doses of antibacterial drugs. A guideline for calculating the breakpoint is reported by the Japanese Society for Chemotherapy Antimicrobial Susceptibility Measurement Method Study Committee.
このガイドラインでは、ＣＬＳＩが設定しているＳ、ＩおよびＲの区分は行わず、ＣＬＳＩでＳに相当する治療効果が期待できるＭＩＣが記載されている。また、患者背景より投与可能な薬剤量を定め、独自のブレイクポイントを設定することも考慮されている。なお、いずれの方法においても、有効な治療方針の策定の指針となっている。 This guideline describes an MIC that does not classify S, I, and R set by CLSI and can expect a therapeutic effect equivalent to S in CLSI. In addition, it is also considered to determine the amount of drug that can be administered from the patient background and to set a unique breakpoint. Each method is a guideline for formulating an effective treatment policy.
一方、長年にわたって抗菌薬を多用してきた結果、抗菌薬に対する耐性菌が多く出現した。更に、近年、医療現場で問題になっている多剤耐性菌が出現し、ほとんどの抗菌薬のＭＩＣが耐性を示している。それゆえ、多剤耐性菌が起因となっている感染症に対して単剤の抗菌薬による治療の効果はほとんど期待できず、有効な治療方法が無いという問題が生じている。 On the other hand, as a result of many use of antibacterial drugs for many years, many resistant bacteria to the antibacterial drugs have appeared. Furthermore, in recent years, multidrug-resistant bacteria that have become a problem in the medical field have appeared, and MICs of most antibacterial drugs have shown resistance. Therefore, the effect of treatment with a single antibacterial agent can hardly be expected for infectious diseases caused by multidrug-resistant bacteria, and there is a problem that there is no effective treatment method.
この状況の中、これらの多剤耐性菌に対して単剤では治療の効果が期待できない抗菌薬も、同様に単剤では治療の効果が期待できない別の抗菌薬と併用することによって治療効果を発揮する組み合わせがあることが知られている。それゆえ、医療現場において、多剤耐性菌に起因する感染症に対して、このような抗菌薬の併用による治療の実施が望まれている。 In this situation, antibacterial drugs that cannot be expected to be effective with a single agent against these multidrug-resistant bacteria can be treated with other antibacterial agents that cannot be expected to be effective with a single agent. It is known that there are combinations that demonstrate. Therefore, in the medical field, it is desired to carry out treatment by using such antibacterial agents in combination for infectious diseases caused by multidrug-resistant bacteria.
しかし、この抗菌薬の併用治療を行うには、予め薬剤感受性検査による抗菌活性の確認が必要である。一般的には、この抗菌薬の併用による効果の適否を判定するために、微量液体希釈法によるチェッカーボード法（非特許文献４）が行われている。 However, in order to perform this antibacterial combination treatment, it is necessary to confirm the antibacterial activity by a drug sensitivity test in advance. In general, a checkerboard method (Non-Patent Document 4) based on a micro liquid dilution method is performed in order to determine whether or not the effect of the combined use of the antibacterial drug is appropriate.
この微量液体希釈法によるチェッカーボード法は、２種類の抗菌薬の２倍連続希釈系列を各々混合してそれらの抗菌活性の有無を調べ、その結果からＦＩＣ係数（ｆｒａｃｔｉｏｎａｌ ｉｎｈｉｂｉｔｏｒｙ ｃｏｎｃｅｎｔｒａｔｉｏｎ ｉｎｄｅｘ）を算定して抗菌薬の併用による効果を評価する。 In this checkerboard method based on the micro liquid dilution method, two-fold serial dilution series of two kinds of antibacterial drugs are mixed and examined for their antibacterial activity, and the FIC coefficient (fractional inhibitory index) is calculated from the results. To evaluate the effect of combined use of antibacterial drugs.
抗菌薬の併用による効果は、期待される相乗的もしくは相加的な抗菌活性の上昇の他に、併用する抗菌薬同士の拮抗作用によって期待された抗菌活性が得られない場合があることが知られている。それゆえ、複数種類の抗菌薬を併用する治療を行う際には、微量液体希釈法によるチェッカーボード法によって得られたＦＩＣ係数を基に、抗菌薬の併用効果を確認する必要がある。 In addition to the expected synergistic or additive increase in antibacterial activity, the antibacterial activity expected from the combined use of antibacterial agents may not be achieved. It has been. Therefore, when performing treatment using a combination of a plurality of types of antibacterial drugs, it is necessary to confirm the combined effect of the antibacterial drugs based on the FIC coefficient obtained by the checkerboard method using the micro liquid dilution method.
この微量液体希釈法によるチェッカーボード法を実施するための一般的な方法としては、９６穴マイクロプレートの各々のウェルに各濃度の抗菌薬の混合物と被験菌を注入し、各々のウェルでの抗菌活性の有無を調べることが知られている。しかし、２種類の抗菌薬の組み合わせは多いため、１枚の９６穴マイクロプレートを使用して、１菌株に対する抗菌薬の１通りの組み合わせしか併用効果の評価ができない。 A general method for carrying out the checkerboard method by this micro liquid dilution method is to inject a mixture of antibacterial drugs of each concentration and a test bacterium into each well of a 96-well microplate, and antibacterial in each well. It is known to check for activity. However, since there are many combinations of two types of antibacterial agents, only one combination of antibacterial agents for one strain can be evaluated using one 96-well microplate.
しかし、感染症の治療に用いる有効な組み合わせの抗菌薬を選択するためには、多種類の抗菌薬の組み合わせを検討する必要がある。それゆえ、微量液体希釈法によるチェッカーボード法を用いて抗菌薬の併用効果を評価する場合には、検討する組み合わせと同数の９６穴マイクロプレートを用いて試験を実施し、その成績を解析する必要があり、適確な試験結果を得るために相当な時間と費用を費やすこととなる。そのため、この検査を頻繁に実施することは容易なことではない。 However, in order to select an effective combination of antibacterial agents for use in the treatment of infectious diseases, it is necessary to consider combinations of various types of antibacterial agents. Therefore, when evaluating the combined effect of antibacterial drugs using the checkerboard method based on the micro liquid dilution method, it is necessary to conduct tests using the same number of 96-well microplates as the combination under consideration and analyze the results. There is a considerable amount of time and money to obtain accurate test results. Therefore, it is not easy to perform this inspection frequently.
また、微量液体希釈法によるチェッカーボード法を実施する目的は、抗菌薬の組み合わせによる治療効果の確認より、むしろ、抗菌薬の組み合わせによる相乗、相加効果もしくは拮抗作用の情報を得る、いわば薬理学的な効果を確認することにある。 The purpose of the checkerboard method by the micro liquid dilution method is not to confirm the therapeutic effect by the combination of antibacterial agents, but to obtain information on synergy, additive effect or antagonism by the combination of antibacterial agents. It is to confirm the effect.
感染症の治療において抗菌薬を併用して用いることの重要性が増している中、有効な抗菌薬の組み合わせを選択するための薬剤感受性検査法の実施が必須となっている。しかし、現在行われている微量液体希釈法によるチェッカーボード法では、適確な試験結果を得るために相当な時間と費用を費やすこととなる。 As the importance of using antibacterial agents in combination in the treatment of infectious diseases is increasing, it is essential to conduct drug susceptibility testing methods to select effective antibacterial drug combinations. However, the current checkerboard method using a micro liquid dilution method requires a considerable amount of time and money to obtain an accurate test result.
本発明はこれらの点を鑑み、感染症の治療における有効な抗菌薬の組み合わせを選定する際に、簡易にかつ経済的に抗菌薬併用効果を判定する薬剤感受性検査法を提供するものである。 In view of these points, the present invention provides a drug sensitivity test method for easily and economically determining an antibacterial combination effect when selecting an effective combination of antibacterial agents in the treatment of infectious diseases.
本発明は、広範囲の２倍連続希釈系列の薬剤濃度で試験を実施していた微量液体希釈法によるチェッカーボード法の煩雑さを改め、定められた薬剤濃度として単剤での抗菌薬の治療効果を判断する際に一般的に用いる薬剤濃度を用いて、抗菌薬の併用による効果を評価することを特徴とする。 The present invention revises the complexity of the checkerboard method based on the micro liquid dilution method, which has been tested with a wide range of 2-fold serial dilution series of drug concentrations, and the therapeutic effect of an antibacterial agent alone as a defined drug concentration It is characterized in that the effect of the combined use of antibacterial drugs is evaluated using the drug concentration generally used when judging the above.
その評価方法として、例えばＣＬＳＩのガイドラインのＳ、Ｉ、Ｒの濃度、日本化学療法学会のガイドラインのブレイクポイントおよびその近傍の濃度等、抗菌薬の有効性の判断基準となる濃度全てもしくはその一部を用いて、微量液体希釈法によるチェッカーボード法と同等の試験を行うことが、効率良くかつ安価に抗菌薬の併用による治療効果の有効な情報が得られると想到した。 As the evaluation method, for example, the concentrations of S, I, and R in the CLSI guidelines, the breakpoints in the guidelines of the Japanese Society of Chemotherapy and the concentration in the vicinity thereof, etc. It was thought that effective information on the therapeutic effect of the combined use of antibacterial drugs can be obtained efficiently and inexpensively by performing a test equivalent to the checkerboard method by the micro liquid dilution method.
そして、本発明者らは、多剤耐性菌に対する抗菌薬の併用療法の効果に関する情報を効率よく得る方法を見いだし、本発明を完成するに至った。 Then, the present inventors have found a method for efficiently obtaining information on the effect of a combination therapy of antibacterial agents against multidrug-resistant bacteria, and have completed the present invention.
すなわち、本発明は以下の構成からなる。
（１）２もしくはそれ以上の、抗菌薬に対する薬剤感受性および／または薬剤耐性の判断基準となる１乃至３の薬剤濃度を用いて２もしくはそれ以上の抗菌薬の併用効果を試験する薬剤感受性検査法を組み合わせた、薬剤感受性検査キット
（２）前記薬剤濃度が日本化学療法学会抗菌薬感受性測定法検討委員会報告により算出されたブレイクポイント近傍の濃度である、（１）記載の薬剤感受性検査キット
（３）前記薬剤濃度がＣｌｉｎｉｃａｌ ａｎｄ Ｌａｂｏｒａｔｏｒｙ Ｓｔａｎｄａｒｄｓ Ｉｎｓｔｉｔｕｔｅ（ＣＬＳＩ）のガイドライン記載のＳｕｓｃｅｐｔｉｂｉｌｉｔｙ（Ｓ：感性）、Ｉｎｔｅｒｍｅｄｉａｔｅ（Ｉ：中間）、Ｒｅｓｉｓｔａｎｃｅ（Ｒ：耐性）の中の濃度である、（１）記載の薬剤感受性検査キット
（４）液体培地を用いて被検菌を増殖させることを特徴とする、（１）〜（３）記載の薬剤感受性検査キット
（５）マイクロプレートを用いて被検菌を増殖させることを特徴とする、（１）〜（４）記載の薬剤感受性検査キット
（６）前記マイクロプレートが９６穴マイクロプレートである、（１）〜（５）記載の薬剤感受性検査キット
That is, the present invention has the following configuration.
(1) A drug sensitivity test method for testing the combined effect of two or more antibacterial agents using 1 to 3 drug concentrations which are criteria for determining drug sensitivity and / or drug resistance to two or more antibacterial agents. (2) The drug sensitivity test kit according to (1), wherein the drug concentration is a concentration in the vicinity of a breakpoint calculated by a report of the Japanese Society for Chemotherapy Antimicrobial Sensitivity Measurement Method Study Committee ( 3) The drug concentration is a concentration among Susceptibility (S: sensitivity), Intermediate (I: intermediate), and Resistance (R: resistance) as described in the guidelines of the Clinical and Laboratory Standards Institute (CLSI). drug susceptibility testing kit (4) Wherein the growing test fungus using somatic medium, and wherein the growing test fungus using (1) to (3), wherein the drug sensitivity test kit (5) a microplate, (1)-(4) drug sensitivity test kit (6) The drug sensitivity test kit according to (1)-(5), wherein the microplate is a 96-well microplate.
複数の抗菌薬による併用療法の治療効果を予測する場合、試験方法として主に微量液体希釈法によるチェッカーボード法が行われている。この試験方法は、例えば２種類の抗菌薬の併用療法の治療効果を予測するために実施する時、各々の抗菌薬の２倍連続希釈系列を９濃度評価するならば、合計８１種類の組み合わせを評価しなければならない。これに比較して、本発明の方法を用いれば、各々の抗菌薬の最大３濃度を評価しても、合計９種類の組み合わせを評価するに過ぎない。 When predicting the therapeutic effect of a combination therapy using a plurality of antibacterial agents, a checkerboard method based on a micro liquid dilution method is mainly used as a test method. For example, when this test method is carried out to predict the therapeutic effect of a combination therapy of two types of antibacterial agents, if 9 concentrations of a 2-fold serial dilution series of each antibacterial agent are evaluated, a total of 81 types of combinations will be obtained. Must be evaluated. Compared to this, if the method of the present invention is used, even if the maximum three concentrations of each antibacterial drug are evaluated, only a total of nine types of combinations are evaluated.
さらに、試験器具として９６穴マイクロプレートを用いた場合、微量液体希釈法によるチェッカーボード法では抗菌薬の１通りの組み合わせしか評価できないのに対し、本発明の方法では抗菌薬の１０通りの組み合わせが評価できる。すなわち、本発明の実施により、効率良くかつ安価に抗菌薬の併用による治療効果の有効な情報を得ることが可能となる。 Furthermore, when a 96-well microplate is used as a test instrument, only one combination of antibacterial drugs can be evaluated by the checkerboard method based on the micro liquid dilution method, whereas ten combinations of antibacterial drugs can be evaluated by the method of the present invention. Can be evaluated. That is, by implementing the present invention, it is possible to obtain effective information on the therapeutic effect of the combined use of antibacterial agents efficiently and inexpensively.
本発明内容の微量液体希釈法を９６穴マイクロプレートに適用する場合、併用療法の治療効果を予測したい被験菌の標準株に対する２種類の抗菌薬のＳ、ＩおよびＲの３濃度を組み合わせた合計９ウェルを用意し、各々のウェルに被験菌を接種して培養し、ウェル内での被験菌の発育の有無を調べることにより、２種類の抗菌薬による併用療法の治療効果の情報が得られる。この試験系において、Ｒの濃度は元来治療効果が期待出来ない濃度であるため、ＳおよびＩの２濃度で試験を行っても同等の試験成績が得られるので、４ウェルでも試験の実施が可能である。 When the micro liquid dilution method of the present invention is applied to a 96-well microplate, the total of three concentrations of S, I, and R of two types of antibacterial agents against the standard strain of the test strain for which the therapeutic effect of the combination therapy is to be predicted By preparing 9 wells, inoculating and culturing the test bacteria in each well, and examining the presence or absence of growth of the test bacteria in the wells, information on the therapeutic effect of the combination therapy with two antibacterial drugs can be obtained . In this test system, since the concentration of R is originally a concentration at which a therapeutic effect cannot be expected, even if the test is performed with two concentrations of S and I, the same test results can be obtained, so the test can be performed even in four wells. Is possible.
また、寒天平板希釈法でも、同様に４枚の抗菌薬を添加した寒天平板培地を準備することで試験の実施が可能となる。さらに、寒天平板培地の広さにもよるが、１枚の寒天平板培地で２０菌株以上の被験菌を試験することが可能である。 In addition, even in the agar plate dilution method, the test can be performed by preparing an agar plate medium to which four antibacterial agents are similarly added. Furthermore, depending on the size of the agar plate medium, it is possible to test 20 or more test bacteria on one agar plate medium.
以下に実施例を挙げて本発明を具体的に説明するが、本発明はこれらにより何ら限定されるものではない。 EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
実施例１．微量液体希釈法を用いた多剤耐性緑膿菌に対する有効抗菌薬併用効果の確認
（１）試験用マイクロプレートの調製
抗菌薬は、シプロフロキサシン（ＣＰＦＸ）、メロペネム（ＭＥＰＭ）、セフタジジム（ＣＡＺ）、ピペラシリン（ＰＩＰＣ）、アズトレオナム（ＡＺＴ）、ゲンタマイシン（ＧＭ）、リファンピシン（ＲＥＰ）およびコリスチン（ＣＬ）を用いた。
Example 1. Confirmation of effective antibacterial drug combination effect against multi-drug resistant Pseudomonas aeruginosa using micro liquid dilution method (1) Preparation of test microplate Antibacterial drugs are ciprofloxacin (CPFX), meropenem (MEPM), ceftazidime (CAZ) ), Piperacillin (PIPC), aztreonam (AZT), gentamicin (GM), rifampicin (REP) and colistin (CL).
薬剤濃度を２濃度とし、主にＣＬＳＩに記載されているＩおよびＳの濃度を用いて抗菌薬の１通りの組み合わせ当たり２×２の４ウェルを使用し、９６穴マイクロプレートに配置した。なお、具体的な薬剤濃度は、ＣＰＦＸ（１および２μｇ／ｍＬ）、ＭＥＰＭ（４および８μｇ／ｍＬ）、ＣＡＺ（８および１６μｇ／ｍＬ）、ＰＩＰＣ（１６および３２μｇ／ｍＬ）、ＡＺＴ（８および１６μｇ／ｍＬ）、ＧＭ（４および８μｇ／ｍＬ）、ＲＥＰ（２および４μｇ／ｍＬ）、ならびにＣＬ（０．５および１μｇ／ｍＬ）とした。 The drug concentration was 2 and 2 × 2 4 wells were used for each combination of antibacterials using the I and S concentrations primarily described in CLSI and placed in a 96-well microplate. Specific drug concentrations are CPFX (1 and 2 μg / mL), MEPM (4 and 8 μg / mL), CAZ (8 and 16 μg / mL), PIPC (16 and 32 μg / mL), AZT (8 and 16 μg). / ML), GM (4 and 8 μg / mL), REP (2 and 4 μg / mL), and CL (0.5 and 1 μg / mL).
本実施例での９６穴マイクロプレートの抗菌薬の配置を図１に示す。ＡおよびＢにＣＰＦＸとＭＥＰＭ、ＣＡＺ、ＰＩＰＣおよびＡＺＴの４通りの組み合わせ、ＣＡＺとＡＺＴの１通りの組み合わせ、ならびに対照を、ＣおよびＤにＧＭとＭＥＰＭ、ＣＡＺ、ＰＩＰＣ、ＡＺＴ、ＣＰＦＸおよびＲＥＰの６通りの組み合わせ、ＥおよびＦにＲＥＰとＭＥＰＭ、ＣＡＺ、ＰＩＰＣ、ＡＺＴ、ＣＰＦＸおよびＣＬの６通りの組み合わせ、
ＧおよびＨにＣＬとＭＥＰＭ、ＣＡＺ、ＰＩＰＣ、ＡＺＴ、ＣＰＦＸおよびＧＭの６通りの組み合わせを各々配置した。なお、接種菌の増殖のための培地は、ミュラーヒントンブイヨンを使用した。
The arrangement of the antibacterial agents in the 96-well microplate in this example is shown in FIG. Four combinations of CPFX and MEPM, CAZ, PIPC and AZT, one combination of CAZ and AZT, and control for A and B, and GM and MEPM, CAZ, PIPC, AZT, CPFX and REP for C and
Six combinations of CL and MEPM, CAZ, PIPC, AZT, CPFX and GM were arranged in G and H, respectively. Note that Muller Hinton bouillon was used as a medium for the growth of the inoculum.
（２）試験菌株
多剤耐性緑膿菌（Ｐｓｅｕｄｏｍｏｎａｓ ａｅｒｕｇｉｎｏｓａ）として、保存菌株であるＥＫＮ５１１２およびＥＫＮ７１１６の２株を用いた。これら２株に対する各抗菌薬のＭＩＣを表１に示す。
(2) Test strains Two strains, EKN5112 and EKN7116, which are conserved strains, were used as multidrug-resistant Pseudomonas aeruginosa. Table 1 shows the MIC of each antibacterial drug against these two strains.
（３）試験法
ＣＬＳＩの微量液体希釈法に準拠して、試験菌を各ウェルに５×１０５ｃｏｌｏｎｙ ｆｏｒｍｉｎｇ ｕｎｉｔｓ（ＣＦＵ）／ｍＬになるように接種し、３５℃で１８〜２４時間培養後、各ウェルの発育の有無を観察した。
(3) Test method In accordance with the micro liquid dilution method of CLSI, inoculate each well with 5 × 10 5 colony forming units (CFU) / mL and incubate at 35 ° C. for 18 to 24 hours. The presence or absence of growth in each well was observed.
（４）結果
使用した８種の抗菌薬の濃度はいずれも試験菌株のＭＩＣより低いため、ほとんどのウェルで発育が阻害されることはなかった。しかし、ＥＫＮ５１１２では、ＧＭ（８）＋ＡＺＴ（８）、ＧＭ（８）＋ＡＺＴ（１６）およびＲＥＰ（４）＋ＣＬ（１）のウェルで発育が阻止された（図２）。それゆえ、本菌株に対してはＧＭとＡＺＴおよびＲＥＰとＣＬの併用での効果が認められた。ＥＫＮ７１１６では、ＲＥＰ（２）＋ＣＬ（１）およびＲＥＰ（４）＋ＣＬ（１）のウェルで発育が阻止された（図３）。それゆえ、本菌株に対してはＲＥＰとＣＬの併用での効果が認められた。このように、９６穴マイクロプレート１枚で８薬剤の２３通りの組み合わせの併用効果を試験することができた。
(4) Results Since the concentrations of the 8 antimicrobial agents used were all lower than the MIC of the test strain, growth was not inhibited in most wells. However, in EKN5112, the growth was blocked in the wells of GM (8) + AZT (8), GM (8) + AZT (16) and REP (4) + CL (1) (FIG. 2). Therefore, the effect of the combined use of GM and AZT and REP and CL was recognized against this strain. EKN7116 inhibited growth in wells of REP (2) + CL (1) and REP (4) + CL (1) (FIG. 3). Therefore, the effect of the combined use of REP and CL was recognized against this strain. Thus, it was possible to test the combined effect of 23 combinations of 8 drugs on one 96-well microplate.
実施例２．寒天平板希釈法を用いた多剤耐性緑膿菌に対する有効抗菌薬併用効果の確認
（１）試験用寒天培地の調製
抗菌薬は、シプロフロキサシン（ＣＰＦＸ）、メロペネム（ＭＥＰＭ）、セフタジジム（ＣＡＺ）、ピペラシリン（ＰＩＰＣ）、アズトレオナム（ＡＺＴ）、ゲンタマイシン（ＧＭ）、リファンピシン（ＲＥＰ）およびコリスチン（ＣＬ）を用いた。
Example 2 Confirmation of effective antibacterial combination effect against multidrug resistant Pseudomonas aeruginosa using agar plate dilution method (1) Preparation of test agar medium Antibiotics are ciprofloxacin (CPFX), meropenem (MEPM), ceftazidime (CAZ) ), Piperacillin (PIPC), aztreonam (AZT), gentamicin (GM), rifampicin (REP) and colistin (CL).
薬剤濃度を２濃度とし、主にＣＬＳＩに記載されているＩおよびＳの濃度を用いて抗菌薬の１通りの組み合わせ当たり２×２の４枚の寒天平板を作成した。なお、具体的な薬剤濃度は、ＣＰＦＸ（１および２μｇ／ｍＬ）、ＭＥＰＭ（４および８μｇ／ｍＬ）、ＣＡＺ（８および１６μｇ／ｍＬ）、ＰＩＰＣ（１６および３２μｇ／ｍＬ）、ＡＺＴ（８および１６μｇ／ｍＬ）、ＧＭ（４および８μｇ／ｍＬ）、ＲＥＰ（２および４μｇ／ｍＬ）、ならびにＣＬ（０．５および１μｇ／ｍＬ）とした。 The concentration of the drug was set to 2, and 4 × 2 agar plates of 2 × 2 were prepared for each combination of antibacterial agents, mainly using the concentrations of I and S described in CLSI. Specific drug concentrations are CPFX (1 and 2 μg / mL), MEPM (4 and 8 μg / mL), CAZ (8 and 16 μg / mL), PIPC (16 and 32 μg / mL), AZT (8 and 16 μg). / ML), GM (4 and 8 μg / mL), REP (2 and 4 μg / mL), and CL (0.5 and 1 μg / mL).
抗菌薬の組み合わせは、ＣＰＦＸとＭＥＰＭ、ＣＰＦＸとＣＡＺ、ＣＰＦＸとＰＩＰＣ、ＣＰＦＸとＡＺＴ、ＣＰＦＸとＧＭ、ＣＰＦＸとＲＥＰ、ＣＰＦＸとＣＬ、ＧＭとＭＥＰＭ、ＧＭとＣＡＺ、ＧＭとＰＩＰＣ、ＧＭとＡＺＴ、ＧＭとＲＥＰ、ＧＭとＣＬ、ＲＥＰとＭＥＰＭ、ＲＥＰとＣＡＺ、ＲＥＰとＰＩＰＣ、ＲＥＰとＡＺＴ、ＲＥＰとＣＬ、ＣＬとＭＥＰＭ、ＣＬとＣＡＺ、ＣＬとＰＩＰＣ、ＣＬとＡＺＴおよびＣＡＺとＡＺＴの合計２３通りの組み合わせを用いた。また、対照として薬剤未添加の培地も用いた。なお、培地は、ミュラーヒントン寒天培地を使用した。 Combinations of antibacterial drugs include CPFX and MEPM, CPFX and CAZ, CPFX and PIPC, CPFX and AZT, CPFX and GM, CPFX and REP, CPFX and CL, GM and MEPM, GM and CAZ, GM and PIPC, GM and AZT, GM and REP, GM and CL, REP and MEPM, REP and CAZ, REP and PIPC, REP and AZT, REP and CL, CL and MEPM, CL and CAZ, CL and PIPC, CL and AZT, CAZ and AZT 23 Street combinations were used. As a control, a medium without any drug was also used. As a medium, Mueller Hinton agar medium was used.
（２）試験菌株
実施例１と同様に多剤耐性緑膿菌（Ｐｓｅｕｄｏｍｏｎａｓ ａｅｒｕｇｉｎｏｓａ）として、保存菌株であるＥＫＮ５１１２とＥＫＮ７１１６の２株を用いた。
(2) Test strain As in Example 1, two strains, EKN5112 and EKN7116, which are conserved strains, were used as multidrug-resistant Pseudomonas aeruginosa.
（３）試験法
ＣＬＳＩの寒天平板希釈法に準拠して、１×１０７ＣＦＵ／ｍＬの濃度になるように試験菌の懸濁液を調製し、専用の接種器を使用して平板培地上に３μＬを接種し、３５℃で１８〜２４時間培養後、接種した菌の発育の有無を観察した。この方法では、培地一枚あたり３２〜３６菌株の試験が可能である。
(3) Test method Based on the CLSI agar plate dilution method, prepare a suspension of test bacteria to a concentration of 1 × 10 7 CFU / mL, and use a special inoculator on the plate medium. 3 μL was inoculated and cultured at 35 ° C. for 18-24 hours, and then the presence or absence of growth of the inoculated bacteria was observed. In this method, 32 to 36 strains can be tested per medium.
（４）結果
使用した８種の抗菌薬の濃度はいずれも試験菌株のＭＩＣより低いため、ほとんどの培地で発育が認められた。しかし、ＥＫＮ５１１２では、ＧＭ（８）＋ＡＺＴ（８）、ＧＭ（８）＋ＡＺＴ（１６）およびＲＥＰ（４）＋ＣＬ（１）の培地で発育が阻止された。それゆえ、本菌株に対してはＧＭとＡＺＴおよびＲＥＰとＣＬの併用での効果が認められた。ＥＫＮ７１１６では、ＲＥＰ（２）＋ＣＬ（１）およびＲＥＰ（４）＋ＣＬ（１）の培地で発育が阻止された。それゆえ、本菌株に対してはＲＥＰとＣＬの併用での効果が認められた。
(4) Results Since the concentrations of the eight antibacterial agents used were all lower than the MIC of the test strain, growth was observed in most culture media. However, in EKN5112, the growth was inhibited in the medium of GM (8) + AZT (8), GM (8) + AZT (16), and REP (4) + CL (1). Therefore, the effect of the combined use of GM and AZT and REP and CL was recognized against this strain. In EKN7116, growth was inhibited in REP (2) + CL (1) and REP (4) + CL (1) media. Therefore, the effect of the combined use of REP and CL was recognized against this strain.
多剤耐性菌に起因する感染症を治療するため、複数の抗菌薬の併用療法を行う際に、使用する抗菌薬を選定するための薬剤感受性検査法を提供する。また、この薬剤感受性検査法のキットを製造し、医療機関の検査室等での簡便な検査の実施に寄与する。 Provided is a drug sensitivity test method for selecting an antibacterial agent to be used when a combination therapy of a plurality of antibacterial agents is performed in order to treat an infection caused by a multidrug resistant bacterium. In addition, this drug sensitivity test method kit will be manufactured to contribute to the implementation of simple tests in medical laboratories.
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