JP2000060595A - Detection of vancomycin-resistant enterococcus and detection medium - Google Patents
Detection of vancomycin-resistant enterococcus and detection mediumInfo
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- JP2000060595A JP2000060595A JP10250416A JP25041698A JP2000060595A JP 2000060595 A JP2000060595 A JP 2000060595A JP 10250416 A JP10250416 A JP 10250416A JP 25041698 A JP25041698 A JP 25041698A JP 2000060595 A JP2000060595 A JP 2000060595A
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、バンコマイシン耐
性腸球菌（Vancomycin-resistant Enterococci、ＶＲ
Ｅ）の検出を行うと同時に、該腸球菌のvanA、ｖanB、
ｖanC遺伝子により決定されるバンコマイシン耐性タイ
プを識別する方法に関する 。TECHNICAL FIELD The present invention relates to vancomycin resistance.
Enterococci (VR)
E) is detected and at the same time, the enterococcal vanA, vanB,
Vancomycin-resistant Thailand determined by the vanC gene
On how to identify a group .
【０００２】[0002]
【従来の技術】ヨーロッパでグリコペプタイド系抗生物
質であるアボパルシンが家畜の飼料に添加され、そこか
らバンコマイシン耐性腸球菌が出現し、ヨーロッパ全体
から全世界に蔓延していったことは広く知られている。
バンコマイシン耐性腸球菌に有効な抗生物質は現時点で
は発見されていないため、感染した場合は早期に発見し
て適切な治療を施す必要がある。It is widely known that in Europe, a glycopartide antibiotic, avoparcin, was added to the feed of livestock, and vancomycin-resistant enterococci emerged from it and spread from all over Europe to the whole world. There is.
Antibiotics effective against vancomycin-resistant enterococci have not been discovered at this time, so if infected, it is necessary to detect it early and provide appropriate treatment.
【０００３】バンコマイシ ン耐性腸球菌の耐性はvanA、
VanB、VanC遺伝子群によってコードされている1)。バン
コマイシン耐性腸球菌の検出方法としては、バンコマイ
シン６μｇ／ｍｌ含有のBile esculine azide agar（Di
fco）、バンコマイシン6μｇ／ｍｌ含有のBrain Heart
infusion agarおよびバンコマイシン８μｇ／ｍｌ含有
のEnterococcosel agar（BBL）等の培地を用いる方法
があり、これらの培地は既に市販されている。しかしな
がら、これらの方法では、バンコマイシンに軽度耐性を
示すバンコマイシン耐性腸球菌（vanB、vanC遺伝子によ
る耐性）は検出されない場合がある。また、バンコマイ
シン耐性腸球菌に感染した場合、その耐性度（vanA、va
nB、vanC遺伝子による耐性）に応じた治療を行う必要が
あるが、これらの方法では、vanA、vanB、vanC 遺伝子
による耐性の発現型の違いを確認できない。Bangkomai Resistance to enterococcus is vanA,
Encoded by the VanB and VanC genes1). Van
As a method for detecting comycin resistant enterococci,
Bile esculine azide agar (Di containing 6 μg / ml of syn
fco), Brain Heart containing vancomycin 6 μg / ml
Contains infusion agar and vancomycin 8 μg / ml
Method using a culture medium such as Enterococcosel agar (BBL)
, And these media are already on the market. But
However, these methods produce mild resistance to vancomycin.
Vancomycin-resistant enterococci (according to the vanB and vanC genes
Resistance) may not be detected. Also, Bangkomai
When infected with syn-resistant enterococci, their degree of resistance (vanA, va
nB, vanC gene resistance)
However, in these methods, the vanA, vanB, vanC genes
It is not possible to confirm the difference in the phenotype of resistance due to.
【０００４】バンコマイシン耐性腸球菌の耐性メカニズ
ムは、既にCouvalin等によって詳細に検討されてい
る2)。vanA遺伝子保有の腸球菌は、バンコマイシンとテ
イコプラニンに対して構成型の高度耐性を示すが、vanB
遺伝子保有の腸球菌はバンコマイシンに対して誘導型の
中程度耐性を示し、テイコプラニンに対しては非誘導型
の中程度耐性を示す3)。また、vanC遺伝子保有の腸球菌
はバンコマイシンとテイコプラニンに対して構成型の軽
度耐性を示す3)。以上のことから、vanA、vanB、vanC各
遺伝子保有菌株は、これら抗菌剤に対する感受性が違
い、耐性誘導も違うことが解る。The resistance mechanism of vancomycin-resistant enterococci has already been examined in detail by Couvalin et al. 2) . Enterococci carrying the vanA gene show constitutive high resistance to vancomycin and teicoplanin, but vanB
Gene-carrying enterococci show inducible moderate resistance to vancomycin and non-inducible moderate resistance to teicoplanin 3) . Also, enterococci carrying the vanC gene show constitutive mild resistance to vancomycin and teicoplanin 3) . From the above, it can be seen that the vanA, vanB, and vanC gene-carrying strains have different susceptibility to these antibacterial agents and different resistance induction.
【０００５】[0005]
【発明が解決しようとする課題】本発明の目的は、従来
法では充分に検出できなかった比較的耐性度の低いvanB
およびvanC保有バンコマイシン耐性腸球菌をも検出可能
な方法を提供することである。本発明の他の目的は、煩
雑なＰＣＲ等で遺伝子自体を検出することなく、従来法
では全く検出、分類できなかったvanA、vanB、vanC遺伝
子型耐性のバンコマイシン耐性腸球菌の検出と分類をす
る方法を提供することである。本発明のさらに他の目的
は臨床検査室（細菌検査室）の従来の検査技術で充分対
応できるバンコマイシン耐性腸球菌の検出と識別方法を
提供することである。DISCLOSURE OF THE INVENTION The object of the present invention is to obtain vanB having a relatively low tolerance which could not be sufficiently detected by the conventional method.
And to provide a method capable of detecting vancomycin-resistant enterococci harboring vanC. Another object of the present invention is to detect and classify vanA, vanB, vanC genotype-resistant vancomycin-resistant enterococci that could not be detected or classified by conventional methods without detecting the gene itself by complicated PCR or the like. Is to provide a method. Still another object of the present invention is to provide a method for detecting and identifying vancomycin-resistant enterococci which can be sufficiently dealt with by conventional testing techniques in clinical laboratories (bacterial testing laboratories).
【０００６】[0006]
【課題を解決するための手段】上記目的を達成するべ
く、本発明者は鋭意検討を行い、バンコマイシン耐性を
獲得している腸球菌に村して、グリコペプタイド系抗生
物質の中で、構成型の耐性を示すグリコペプタイド系抗
生物質と、誘導型耐性を示すグリコペプタイド系抗生物
質を組み合わせることにより、バンコマイシン耐性腸球
菌の検出と、vanA、vanB、VanC遺伝子による耐性の鑑別
が行えると考えた。即ち、バンコマイシン耐性腸球菌
（vanA、vanB、vanC遺伝子保有腸球菌）に対するテイコ
プラニンの各感受性、特にvanB遺伝子に対するバンコマ
イシンの耐性誘導と、vanA、vanB、vanC遺伝子保有腸球
菌の菌株に対するバンコマイシンの感受性を利用するこ
とに想到し、本発明を完成するに至った。[Means for Solving the Problems] In order to achieve the above-mentioned object, the present inventor has conducted diligent studies and found that enterococci which have acquired vancomycin resistance are selected as a constitutive type among glycopeptide antibiotics. It was considered that the combination of a glycopeptide antibiotic showing resistance to Pseudomonas aeruginosa and a glycopeptide antibiotic showing inducible resistance could detect vancomycin-resistant enterococci and distinguish resistance by the vanA, vanB, and VanC genes. That is, the teicoplanin susceptibility to vancomycin-resistant enterococci (vanA, vanB, and vanC gene-carrying enterococci), particularly vancomycin resistance induction to the vanB gene, and vancomycin susceptibility to vanA, vanB, and vanC gene-carrying enterococcus strains are used. As a result, the present invention has been completed.
【０００７】即ち、本発明はバンコマイシン耐性腸球菌
に対して構成型の耐性を示すグリコペプタイド系抗生物
質を含有しかつバンコマイシン耐性腸球菌が生育可能な
培地表面に菌を塗抹後、該菌塗抹表面の一部はバンコマ
イシン耐性腸球菌に対して誘導型耐性を示すグリコペプ
タイド系抗生物質存在下、他部は不存在下に培養するこ
とを特徴とするバンコマイシン耐性腸球菌の検出方法、
及びこのようにして培養した菌の生育状態によりバンコ
マイシン耐性腸球菌のバンコマイシン耐性遺伝子型を識
別する方法である。構成型の耐性を示すグリコペプタイ
ド系抗生物質としてはテイコプラニンが好ましく、誘導
型耐性を示すグリコペプタイド系抗生物質としてはバン
コマイシンが好ましい。That is, according to the present invention, a medium containing a glycopeptide antibiotic showing constitutive resistance to vancomycin-resistant enterococci and capable of growing vancomycin-resistant enterococci is smeared with the bacteria, and the surface of the bacteria is smeared. Part of the presence of a glycopeptide antibiotic showing inducible resistance to vancomycin-resistant enterococci, the other part is a method for detecting vancomycin-resistant enterococci characterized by culturing in the absence,
And a method for distinguishing the vancomycin-resistant genotype of vancomycin-resistant enterococcus by the growth state of the thus-cultivated bacteria. Teicoplanin is preferred as the glycopeptide antibiotic showing constitutive resistance, and vancomycin is preferred as the glycopeptide antibiotic showing inducible resistance.
【０００８】[0008]
【発明の実施の形態】本発明における培地はテイコプラ
ニンを含有するバンコマイシン耐性腸球菌が生育可能な
培地を用いる。テイコプラニン含有培地とは、テイコプ
ラニンを含有し、Enterococciが発育可能な培地であれ
ば市販のものでも独自に作った培地でも差し支えない。
このような培地としては、例えば Brain heart infusio
n agar、Heart infusion agar、Torypto Soy agar、Mul
ler hinton agar、Nutrient agar、血液寒天培地等にテ
イコプラニンを含有させたものが挙げられる。テイコプ
ラニン含有培地に含有するテイコプラニン濃度は、０．
１〜５０μｇ／ｍｌの範囲が好ましく、さらに好ましく
は２〜１５μｇ／ｍｌの範囲である。BEST MODE FOR CARRYING OUT THE INVENTION The medium used in the present invention is a medium which can grow vancomycin-resistant enterococci containing teicoplanin. The teicoplanin-containing medium may be a commercially available medium or an original medium as long as it is a medium containing teicoplanin and capable of developing Enterococci.
Examples of such a medium include Brain heart infusio
n agar, Heart infusion agar, Torypto Soy agar, Mul
Examples include ler hinton agar, nutrient agar, blood agar medium and the like containing teicoplanin. The concentration of teicoplanin contained in the teicoplanin-containing medium was 0.
The range is preferably 1 to 50 μg / ml, more preferably 2 to 15 μg / ml.
【０００９】本発明においてはテイコプラニン含有培地
に塗抹した菌の一部をバンコマイシンが存在する状態に
するには、例えばバンコマイシンを含む媒体を該テイコ
プラニン含有培地に接触されればよい。媒体としては、
含有するバンコマイシンを培地上で拡散できるものであ
ればよく、ペーパー若しくはペーパーディスクが好まし
く使用できる。バンコマイシン含有媒体に含有されるバ
ンコマイシン濃度は、０．１〜１０００μｇ／ｍｌが好
ましく、さらに好ましくは０．１〜５０μｇ／ｍｌであ
る。In the present invention, a part of the bacteria smeared on the teicoplanin-containing medium can be brought into the state in which vancomycin is present by, for example, bringing a medium containing vancomycin into contact with the teicoplanin-containing medium. As a medium,
Any material can be used as long as it can diffuse the contained vancomycin on the medium, and paper or a paper disk can be preferably used. The vancomycin concentration contained in the vancomycin-containing medium is preferably 0.1 to 1000 μg / ml, more preferably 0.1 to 50 μg / ml.
【００１０】本発明の方法によりバンコマイシン耐性腸
球菌を検出し、その耐性遺伝子型を識別するには、具体
的には例えば以下のようにする。まず、テイコプラニン
含有培地でプレートを作成し、試料である培養した菌液
をプレート表面に塗抹する。菌液の濃度は特に制限はな
いが、通常ＯＤ５７８ｎｍで０．０５〜３．０である。
その上にバンコマイシン含有ペーパーディスクを置いて
培養し、培養菌の生育状態を観察する。培養時間は、va
nAおよびvanB保有を確認する場合は１６〜４８時間、Va
nC遺伝子保有を確認する場合は、vanA、vanBの場合より
も長時間で約１８〜７２時間培養するとよい。培養温度
は２７〜４２℃が好ましい。In order to detect vancomycin-resistant enterococci by the method of the present invention and identify the resistant genotype, specifically, for example, the following is carried out. First, a plate is prepared using a teicoplanin-containing medium, and a sample of cultured bacterial solution is smeared on the plate surface. The concentration of the bacterial solution is not particularly limited, but it is usually 0.05 to 3.0 at OD578 nm.
A vancomycin-containing paper disk is placed on it and cultured, and the growth state of the culture is observed. The incubation time is va
16-48 hours to confirm possession of nA and vanB, Va
When confirming the possession of the nC gene, it is advisable to carry out the culture for about 18 to 72 hours, which is longer than that for vanA and vanB. The culture temperature is preferably 27 to 42 ° C.
【００１１】VanA遺伝子による耐性の場合は構成的な高
度耐性であるため、全面に菌が生育するが、vanB遺伝子
の場合はバンコマイシンによる誘導耐性であるため、デ
ィスク周辺にのみ生育が観察される。VanC遺伝子の場合
には耐性は誘導されず、逆にバンコマイシンによる生育
阻害作用として阻止円が形成される。バンコマイシン感
性菌の場合はいずれの培地でも生育しない。[0012] In the case of resistance by the VanA gene, the fungus grows on the entire surface because of the constitutive high resistance, whereas in the case of the vanB gene, the resistance is induced by vancomycin, so that the growth is observed only around the disc. In the case of the VanC gene, resistance is not induced, and conversely a blocking circle is formed as a growth inhibitory action by vancomycin. Vancomycin-sensitive bacteria do not grow on any medium.
【００１２】[0012]
【実施例】以下に本発明を実施例で説明する。
実施例１
＜バンコマイシン耐性腸球菌の検出及びＰＣＲ法との比
較＞以下に試験方法を記す。テイコプラニンを8μｇ／
ｍｌ含有Enterococcosel培地でプレートを作製する。一
夜培養したバンコマイシン耐性腸球菌をＯＤ５７８ｎｍ
で０．３（マックファーランド1）に調整後、滅菌綿棒
を用いて作製した培地表面全面に塗抹する。バンコマイ
シンを０．５、５、５０μｇ含有のペーパーディスクを
作製し、菌を塗抹した培地表面に置く。３７℃で１８時
間培養後、判定する。vanC遺伝子保有の確認は、48時間
後に判定する。試料としてvanA保有バンコマイシン耐性
腸球菌を２５株、vanB保有菌株を５株、vanC保有菌株を
５株、van遺伝子を保有していない３株を使用した。そ
の他に、バンコマイシン（ＶＣＭ）とテイコプラニン
（ＴＥＩＣ）の日本化学療法学会最小発育阻止濃度（Ｍ
ＩＣ）測定方法に従ったＭＩＣ測定とＰＣＲ法を使った
vanA、vanB、vanC遺伝子の検出（P.Courvalin等の方法
参照4))を行い、上記検出方法で得られた結果と比較し
た。EXAMPLES The present invention will be described below with reference to examples. Example 1 <Detection of vancomycin-resistant enterococci and comparison with PCR method> The test methods are described below. Teicoplanin 8 μg /
Make plates in Enterococcosel medium containing ml. Vancomycin-resistant enterococci cultivated overnight were OD578nm
After adjusting to 0.3 (Mac Farland 1), smear it over the entire surface of the prepared medium with a sterile cotton swab. A paper disc containing 0.5, 5, and 50 μg of vancomycin is prepared and placed on the surface of the medium on which the bacteria have been smeared. After culturing at 37 ° C. for 18 hours, determination is made. Confirmation of vanC gene possession is determined after 48 hours. As samples, 25 strains of vanComycin-resistant enterococci having vanA, 5 strains having vanB, 5 strains having vanC, and 3 strains not having van gene were used. In addition, the minimum inhibitory concentration (M) of vancomycin (VCM) and teicoplanin (TEIC) of the Japanese Society of Chemotherapy
IC) MIC measurement according to measurement method and PCR method were used
The vanA, vanB and vanC genes were detected (see the method of P. Courvalin et al. 4) ) and compared with the results obtained by the above detection method.
【００１３】図１にvanA、vanB、VanCタイプに鑑別され
た写真を示す。vanAによる耐性は、構成的な高度耐性で
あるためシャーレ全面に生育してしまう。VanBの場合は
ディスクに含有されるバンコマイシンによって耐性が誘
導されるため、ディスク周辺にのみ生育円が観察され
る。VanCの場合はディスクに含有されるバンコマイシン
によっても耐性は誘導されず、逆にバンコマイシンによ
る生育阻害作用として阻止円が形成される。FIG. 1 shows photographs classified into vanA, vanB, and VanC types. The resistance by vanA grows on the entire surface of the petri dish because of its highly constitutive resistance. In the case of VanB, resistance is induced by vancomycin contained in the disc, so a growth circle is observed only around the disc. In the case of VanC, resistance is not induced by vancomycin contained in the disc, and conversely, an inhibition circle is formed as a growth inhibitory action by vancomycin.
【００１４】表１にvanA保有バンコマイシン耐性E．fae
ciumの結果を示す。表２にvanB保有バンコマイシン耐性
E．faeciumの結果を示す。表３にvanC保有E．gallinari
umの結果を示す。表４にバンコマイシン感性E．faecali
s及びE．faeciumの結果を示す。バンコマイシンとテイ
コプラニンに対するＭＩＣ値が１００μｇ／ｍｌ以上の
菌株は、ＰＣＲで全てvanA遺伝子が確認され、この検出
方法で確認された結果と１００％一致していた。また、
バンコマイシンのＭＩＣ値が１２．５μｇ／ｍｌ以上
で、テイコプラニンのＭＩＣ値が１．５６μｇ／ｍｌ以
下の菌株は、ＰＣＲでvanB遺伝子が確認されたが、この
結果も全て一致していた。更に、バンコマイシンとテイ
コプラニン感受性（ＭＩＣ値で１．５６μｇ／ｍｌ以
下）の菌株は、ＰＣＲでvanC遺伝子が確認されている
が、これもこの検出法方の結果と一致していた。以上の
結果より、本発明の検出方法で鑑別されたvanA、vanB、
vanC遺伝子の結果は、ＰＣＲで確認されたvanA、vanB、
vanC遺伝子の結果と全て一致していた。Table 1 shows vanComycin-resistant E. fae
The result of cium is shown. Table 2 shows vanBomycin resistance with vanB
E. The result of faecium is shown. Table 3 shows E. gallinari
The result of um is shown. Table 4 shows vancomycin-sensitive E. faecali
s and E. The result of faecium is shown. All the strains with MIC values of 100 μg / ml or more for vancomycin and teicoplanin confirmed the vanA gene by PCR, and were 100% in agreement with the results confirmed by this detection method. Also,
The vanB gene was confirmed by PCR in the strains with vancomycin MIC value of 12.5 μg / ml or more and teicoplanin MIC value of 1.56 μg / ml or less, and all the results were also in agreement. Furthermore, the vanC gene was confirmed by PCR in the strains sensitive to vancomycin and teicoplanin (MIC value of 1.56 μg / ml or less), which was also in agreement with the result of this detection method. From the above results, vanA, vanB, distinguished by the detection method of the present invention,
The results of the vanC gene are confirmed by PCR, vanA, vanB,
All were in agreement with the results of the vanC gene.
【００１５】[0015]
【表１】 [Table 1]
【００１６】[0016]
【表２】 [Table 2]
【００１７】[0017]
【表３】 [Table 3]
【００１８】[0018]
【表４】 [Table 4]
【００１９】実施例２
＜市販バンコマイシン耐性腸球菌検出培地との比較＞市
販されているバンコマイシン耐性腸球菌検出培地のEnte
rococcosel培地（バンコマイシン８μｇ／ｍｌ含有）と
Brain Heart Infusion Agar（バンコマイシン６μｇ／
ｍｌ含有）（ＢＨＩＡ）との検出成績の比較を行った。
表５にvanA保有バンコマイシン耐性E．faeciumの市販培
地との検出型の比較結果を示す。vanAによる耐性は、構
成的な高度耐性であるため、何れの培地を用いてもシャ
ーレ全面に生育してしまう。Example 2 <Comparison with a commercially available vancomycin-resistant enterococcal detection medium> A commercially available vancomycin-resistant enterococcal detection medium Ente
rococcosel medium (containing vancomycin 8 μg / ml)
Brain Heart Infusion Agar (vancomycin 6 μg /
(containing ml) (BHIA).
Table 5 shows vanComycin resistant E. The result of comparison of the detection type with the commercial medium of faecium is shown. Since the resistance by vanA is highly constitutive, it grows on the entire surface of the petri dish using any medium.
【００２０】表６にvanB保有バンコマイシン耐性E.faec
iumとの検出型の比較結果を示す。ディスクに含有され
るバンコマイシンによって、vanBの耐性が誘導されるた
め、ディスク周辺にのみ生育円が観察される。バンコマ
イシンによる耐性誘導がなければ、培地に含有されるテ
イコプラニンによって生育は完全に阻害されてしまう。
表７にvanC保有E.gallinariumとの検出型の比較結果を
示す。ディスクに含有されるバンコマイシンによって
も、vanCの耐性は誘導されず、逆にバンコマイシンによ
る生育阻害作用として阻止円が形成される。表８にバン
コマイシン感性E.faecalis及びE.faeciumとの検出型の
比較結果を示す。バンコマイシン感性菌はいずれの培地
でも生育しない。Table 6 shows vanBomycin-resistant E.faec having vanB.
The comparison result of detection type with ium is shown. Since vancomycin contained in the disc induces vanB resistance, a growth circle is observed only around the disc. Without the induction of resistance by vancomycin, the growth is completely inhibited by teicoplanin contained in the medium.
Table 7 shows the results of comparison of detection types with E.gallinarium possessing vanC. Vancomycin contained in the disc also does not induce vanC resistance, and conversely forms an inhibition circle as a growth inhibitory action by vancomycin. Table 8 shows the results of comparison of the detection types with vancomycin-sensitive E.faecalis and E.faecium. Vancomycin-sensitive bacteria do not grow in any medium.
【００２１】[0021]
【表５】 [Table 5]
【００２２】[0022]
【表６】 Disc周辺：Disc周辺にのみ生育円が観察される[Table 6] Around Disc: Growing circles are observed only around Disc
【００２３】[0023]
【表７】 一部生育；菌量が濃い部分のみ生育が確認できる。[Table 7] Partial growth: Growth can be confirmed only in the part with a high bacterial load.
【００２４】[0024]
【表８】 [Table 8]
【００２５】[0025]
【発明の効果】市販培地を用いたバンコマイシン耐性腸
球菌の検出の場合、vanAとvanBはシャーレ全面の生育が
認められ、vanAとvanBの鑑別は行えない。しかし、本発
明の検出培地では、vanAは全面に生育するが、vanBはdi
sc周辺のみの生育円となりvanAとvanBの鑑別が行える。
また、市販培地を用いた場合のvanCの検出は、全面から
一部（菌量の濃い場所）の生育であり、全面生育の場
合、vanA、vanBとの区別もつかない。また、一部生育し
た場合も、菌量が濃すぎたための擬似陽性か、耐性を持
っているための陽性か、区別がつかない場合がある。し
かし、本発明の検出方法であれば、シャーレ全面に生育
し、かつ、バンコマイシン含有disc周辺に阻止円が形成
されるため、vanA、vanBとの明らかな区別が行える。当
然、バンコマイシン感性菌は何れの検出培地を用いても
生育しない（検出されない）。EFFECTS OF THE INVENTION In the case of detecting vancomycin-resistant enterococci using a commercially available medium, vanA and vanB show growth on the entire petri dish, and vanA and vanB cannot be distinguished. However, in the detection medium of the present invention, vanA grows on the entire surface, while vanB di
The growth circle is only around sc, and you can distinguish between vanA and vanB.
In addition, the detection of vanC when using a commercially available medium is a growth from the entire surface to a part (a place where the amount of bacteria is high). In addition, even in the case of partial growth, it may not be possible to distinguish between false positives due to excessive bacterial load or positives due to resistance. However, according to the detection method of the present invention, since it grows on the entire surface of the petri dish and an inhibition circle is formed around the vancomycin-containing disc, it can be clearly distinguished from vanA and vanB. Naturally, vancomycin-susceptible bacteria do not grow (not detected) using any detection medium.
【００２６】本発明のバンコマイシン耐性腸球菌検出方
法は臨床検査室（細菌検査室）の従来の検査技術で充分
対応できる検出方法であり、煩雑雑なＰＣＲ法を行う必
要なくvan遺伝子（vanA、vanB、VanC）の鑑別が行え
る。そのため検査時間が大幅に短縮でき、早期での治療
方法、治療薬の選定が可能になる。更に、バンコマイシ
ン耐性腸球菌の院内感染も重要な問題になっているが、
この検出方法を用いれば院内感染対策を行う上で、早期
の対応が出来るばかりでなく、重要な情報（vanA、van
B、vanc遺伝子の鑑別）も得られる。The method for detecting vancomycin-resistant enterococcus of the present invention is a detection method that can be sufficiently coped with by conventional testing techniques in clinical laboratories (bacterial laboratories), and the van genes (vanA, vanB are not required to perform complicated PCR methods. , VanC) can be distinguished. Therefore, the inspection time can be significantly shortened, and the treatment method and the therapeutic drug can be selected at an early stage. Furthermore, nosocomial infection with vancomycin-resistant enterococci has become an important issue,
When this detection method is used, it is possible not only to take an early action but also to provide important information (vanA, van
(B, vanc gene discrimination) can also be obtained.
【００２７】[0027]
【文献】1)Leclercq R.,E.Derlot,J.Duval,and P.Courv
alin.Plasmid-mediated resistance to vancomycin and
teicoplaninin in Enterococcus faecium. N.Engl.J.M
ed.319:157-161,1988.
2)Courvalin P.Resistance of enterococci to glycope
ptides. Antimicrob.Agent Chemother.34:2291-2296,19
91.
3)Arthur M.,and P.Courvalin. Genetics and mechanis
ms of glycopeptide resistance in Enterococci. Anti
microb. Agents Chemother.37:1563-1571,1993.
4)Clark N.C.,R.C.Cooksey,B.C.Hill, J.M.Swenson,and
F.C.Tenover.Characterization of glycopeptide-resi
stant Enterococci from U.S.hospitals.Antimicrob.Ag
ents Chemother.37:2311-2317,1993.[Reference] 1) Leclercq R., E. Derlot, J. Duval, and P. Courv
alin.Plasmid-mediated resistance to vancomycin and
teicoplaninin in Enterococcus faecium.N.Engl.JM
ed.319: 157-161, 1988. 2) Courvalin P. Resistance of enterococci to glycope
ptides. Antimicrob. Agent Chemother. 34: 2291-2296,19
91.3) Arthur M., and P. Courvalin. Genetics and mechanis
ms of glycopeptide resistance in Enterococci. Anti
microb.Agents Chemother.37: 1563-1571,1993. 4) Clark NC, RCCooksey, BCHill, JMSwenson, and
FCTenover.Characterization of glycopeptide-resi
stant Enterococci from UShospitals.Antimicrob.Ag
ents Chemother. 37: 2311-2317,1993.
【図１】バンコマイシン耐性菌掲遺伝子の相違による菌
の成育の相違を表した写真である。FIG. 1 is a photograph showing a difference in growth of bacteria due to a difference in vancomycin-resistant strain gene.
───────────────────────────────────────────────────── フロントページの続き Ｆターム(参考） 4B029 AA07 BB02 FA01 4B063 QA01 QQ06 QR68 QR69 QR84 QX01 ─────────────────────────────────────────────────── ─── Continued front page F-term (reference) 4B029 AA07 BB02 FA01 4B063 QA01 QQ06 QR68 QR69 QR84 QX01
Claims (11)
型耐性を示すグリコペプタイド系抗生物質を含有しかつ
バンコマイシン耐性腸球菌が生育可能な培地表面に菌を
塗抹後、該菌塗抹表面の一部はバンコマイシン耐性腸球
菌に対して誘導型耐性を示すグリコペプタイド系抗生物
質存在下、他部は不存在下に培養することを特徴とする
バンコマイシン耐性腸球菌の検出方法。1. A medium containing a glycopeptide antibiotic showing constitutive resistance to vancomycin-resistant enterococci and capable of growing vancomycin-resistant enterococci after being smeared with a bacterium, and a part of the bacterium smear surface is A method for detecting vancomycin-resistant enterococci, which comprises culturing in the presence of a glycopeptide antibiotic showing inducible resistance to vancomycin-resistant enterococci and in the absence of other parts.
生物質を含有しかつバンコマイシン耐性腸球菌が生育可
能な培地表面に菌を塗抹後、該菌塗抹表面の一部は誘導
型耐性を示すグリコペプタイド系抗生物質存在下、他部
は不存在下に培養させ、菌の生育状態によりバンコマイ
シン耐性腸球菌のバンコマイシン耐性遺伝子型を識別す
る方法。2. A glycopeptide, which comprises a glycopeptidic antibiotic exhibiting constitutive resistance and is spread on a surface of a medium capable of growing vancomycin-resistant enterococci, and a part of the surface of the bacterial smear shows inducible resistance. A method of culturing in the presence of other antibiotics and in the absence of other parts, and identifying the vancomycin-resistant genotype of vancomycin-resistant enterococci depending on the growth state of the bacterium.
生物質がテイコプラニンであり、誘導型耐性を示すグリ
コペプタイド系抗生物質がバンコマイシンである請求項
１または２の方法。3. The method according to claim 1 or 2, wherein the glycopeptide antibiotic showing constitutive resistance is teicoplanin and the glycopeptide antibiotic showing inducible resistance is vancomycin.
有させて培地表面に置くことにより、菌塗抹培地表面の
一部をバンコマイシンが存在する状態とすることを特徴
とする請求項３の方法。4. The method according to claim 3, wherein vancomycin is contained in a paper disc and placed on the surface of the culture medium, whereby a part of the surface of the bacterial smear medium is brought into the state in which vancomycin is present.
〜５０μｇ／ｍｌである請求項３または４の方法。5. The concentration of teicoplanin in the medium is 0.1.
The method according to claim 3 or 4, wherein the method is about 50 µg / ml.
濃度が０．１〜５０μｇ／ｍｌである請求項４又は５の
方法。6. The method according to claim 4, wherein the concentration of vancomycin in the paper disk is 0.1 to 50 μg / ml.
抗生物質を含有する腸球菌生育可能な培地。7. An enterococci-growing medium containing a glycopeptide antibiotic showing constitutive resistance.
抗生物質がテイコプラニンである請求項７の培地。8. The medium according to claim 7, wherein the glycopeptide antibiotic showing constitutive resistance is teicoplanin.
生物質を含有するペーパーディスク。9. A paper disk containing a glycopeptide antibiotic showing inducible resistance.
抗生物質がバンコマイシンである請求項９のペーパーデ
ィスク。10. The paper disk according to claim 9, wherein the glycopeptide antibiotic showing inducible resistance is vancomycin.
１０のペーパーディスクよりなるバンコマイシン耐性腸
球菌検出および識別キット。11. A kit for detecting and identifying vancomycin-resistant enterococcus, which comprises the medium according to claim 7 or 8 and the paper disc according to claim 9 or 10.
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