JP4083308B2 - Detection method and detection medium for vancomycin-resistant enterococci - Google Patents
Detection method and detection medium for vancomycin-resistant enterococci Download PDFInfo
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Description
【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、ＶＲＥ）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。
【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。
【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（Difco）、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、vanB、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。
【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。
【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。
【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。
【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。
【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 infusion agar、Heart infusion agar、Torypto Soy agar、Muller 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。
【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。
【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、vanAおよびvanB保有を確認する場合は１６〜４８時間、VanC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。
【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】
【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（ＭＩＣ）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。
【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。
【００１４】
表１にvanA保有バンコマイシン耐性E．faecium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．gallinariumの結果を示す。
表４にバンコマイシン感性E．faecalis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。
【００１５】
【表１】
【表２】
【表３】
【表４】
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Enterococcosel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。
【００２０】
表６にvanB保有バンコマイシン耐性E.faecium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。
【００２１】
【表５】
【表６】
【００２３】
【表７】
【００２４】
【表８】
【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disc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）。
【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、vanB、vanc遺伝子の鑑別）も得られる。
【００２７】
【文献】
1)Leclercq R.,E.Derlot,J.Duval,and P.Courvalin.Plasmid-mediated resistance to vancomycin and teicoplaninin in Enterococcus faecium. N.Engl.J.Med.319:157-161,1988.
2)Courvalin P.Resistance of enterococci to glycopeptides. Antimicrob.Agent Chemother.34:2291-2296,1991.
3)Arthur M.,and P.Courvalin. Genetics and mechanisms of glycopeptide resistance in Enterococci. Antimicrob. 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-resistant Enterococci from U.S.hospitals.Antimicrob.Agents Chemother.37:2311-2317,1993.
【図面の簡単な説明】
【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。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for distinguishing vancomycin-resistant types determined by vancomycin-resistant enterococci (VRE) and at the same time determining vancomycin-resistant enterococci (VRE).
[0002]
[Prior art]
It is well known that avopalcin, a glycopeptide antibiotic, was added to livestock feed in Europe, and vancomycin-resistant enterococci emerged from it and spread throughout Europe and around the world. Antibiotics effective against vancomycin-resistant enterococci have not been discovered at this time, so if they are infected, they need to be detected early and treated appropriately.
[0003]
The resistance of vancomycin-resistant enterococci is encoded by the vanA, VanB, and VanC gene families1 ) . As a method for detecting vancomycin-resistant enterococci, media such as Bile esculine azide agar (Difco) containing vancomycin 6 μg / ml, Brain Heart infusion agar containing vancomycin 6 μg / ml, and Enterococcosel agar (BBL) containing vancomycin 8 μg / ml are used. There are methods used, and these media are already commercially available. However, these methods may not detect vancomycin-resistant enterococci (resistance by vanB and vanC genes) that are mildly resistant to vancomycin. In addition, when infected with vancomycin-resistant enterococci, it is necessary to treat according to the degree of resistance (resistance by vanA, vanB, vanC genes), but in these methods, resistance is expressed by vanA, vanB, vanC genes. I cannot confirm the difference in type.
[0004]
Resistance mechanisms of vancomycin-resistant enterococci, 2 that have already been studied in detail by Couvalin etc.). Enterococci possessing vanA gene show constitutive high resistance to vancomycin and teicoplanin, but enterococci possessing vanB gene show inducible moderate resistance to vancomycin and non-induction to teicoplanin 3) Moderate tolerance of the mold. In addition, enterococci possessing vanC gene show constitutive mild resistance to vancomycin and teicoplanin3 ) . From the above, it can be seen that each of the vanA, vanB, and vanC gene-bearing strains has different susceptibility to these antibacterial agents and different resistance induction.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide a method capable of detecting vancomycin-resistant vancomycin-resistant enterococci having a relatively low resistance level that could not be sufficiently detected by conventional methods. Another object of the present invention is to detect and classify vancomycin-resistant enterococci that are resistant to vanA, vanB, and vanC genotypes 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 that can be adequately handled by conventional testing techniques in clinical laboratories (bacteria laboratories).
[0006]
[Means for Solving the Problems]
In order to achieve the above-mentioned object, the present inventor has intensively studied, worked for enterococci that have acquired vancomycin resistance, and among glycopeptide antibiotics, glycopeptide antibiotics exhibiting constitutive resistance And the combination of glycopeptide antibiotics showing inducible resistance, we thought that vancomycin-resistant enterococci could be detected and resistance could be differentiated by vanA, vanB, and VanC genes. That is, teicoplanin susceptibility to vancomycin-resistant enterococci (enterococci possessing vanA, vanB, and vanC genes), in particular, induction of vancomycin resistance to vanB gene and susceptibility of vancomycin to enterococci strains that possess vanA, vanB, and vanC genes. The present invention has been completed.
[0007]
That is, the present invention contains a glycopeptide antibiotic showing constitutive resistance against vancomycin-resistant enterococci and is smeared on the surface of a medium on which vancomycin-resistant enterococci can grow, and then a part of the bacterial smear surface. Is a method for detecting vancomycin-resistant enterococci characterized by culturing in the presence of a glycopeptide antibiotic showing inducible resistance to vancomycin-resistant enterococci and in the absence of other parts, and thus culturing This is a method for discriminating vancomycin-resistant genotypes of vancomycin-resistant enterococci according to the growth state of the bacteria.
Teicoplanin is preferred as a glycopeptide antibiotic exhibiting constitutive resistance, and vancomycin is preferred as a glycopeptide antibiotic exhibiting inducible resistance.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
As the medium in the present invention, a medium capable of growing vancomycin-resistant enterococci containing teicoplanin is used. The teicoplanin-containing medium may be a commercially available medium or an original medium as long as it contains teicoplanin and can grow Enterococci. Examples of such a medium include brain heart infusion agar, heart infusion agar, torypto soy agar, muller hinton agar, nutrient agar, blood agar medium and the like containing teicoplanin. The concentration of teicoplanin contained in the teicoplanin-containing medium is preferably in the range of 0.1 to 50 μg / ml, more preferably in the range of 2 to 15 μg / ml.
[0009]
In the present invention, for example, a medium containing vancomycin may be brought into contact with the teicoplanin-containing medium in order to bring vancomycin into a part of the bacteria smeared on the teicoplanin-containing medium. Any medium can be used as long as it can diffuse the vancomycin contained on the medium, and paper or 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]
In order to detect vancomycin-resistant enterococci by the method of the present invention and identify the resistant genotype, specific examples are as follows.
First, a plate is prepared with a teicoplanin-containing medium, and a cultured bacterial solution as a sample is smeared on the plate surface. The concentration of the bacterial solution is not particularly limited, but is usually 0.05 to 3.0 at OD578 nm. A vancomycin-containing paper disk is placed thereon and cultured, and the growth state of the culture is observed. When confirming vanA and vanB possession, the culture time is 16 to 48 hours, and when confirming VanC gene possession, the culture time is preferably longer than that of vanA and vanB for about 18 to 72 hours. The culture temperature is preferably 27 to 42 ° C.
[0011]
In the case of resistance due to the VanA gene, since it is constitutively high resistance, bacteria grow on the entire surface, whereas in the case of the vanB gene, growth is observed only around the disc because of resistance induced by vancomycin. In the case of the VanC gene, no tolerance is induced, and conversely, a blocking circle is formed as a growth inhibitory action by vancomycin. In the case of vancomycin-sensitive bacteria, it does not grow in any medium.
[0012]
【Example】
Hereinafter, the present invention will be described with reference to examples.
Example 1
<Detection of vancomycin-resistant enterococci and comparison with PCR method>
The test method is described below.
Plates are made in Enterococcosel medium containing 8 μg / ml teicoplanin.
Vancomycin-resistant enterococci cultured overnight are adjusted to 0.3 (Macfarland 1) at OD578 nm and then smeared on the entire surface of the medium prepared using a sterile cotton swab. Paper discs containing vancomycin 0.5, 5, and 50 μg are prepared and placed on the surface of the medium smeared with bacteria. After 18 hours of incubation at 37 ° C., determination is made. Confirmation of vanC gene possession is determined 48 hours later.
As a sample, 25 vancomycin-resistant enterococci having vanA, 5 strains having vanB, 5 strains having vanC, and 3 strains having no van gene were used.
In addition, vanAmycin (VCM) and teicoplanin (TEIC) detection of vanA, vanB, and vanC genes using the PCR method and MIC measurement according to the Japanese Society of Chemotherapy Minimum Inhibitory Concentration (MIC) (P. Courvalin et al. 4) ), and compared with the results obtained by the above detection method.
[0013]
Fig. 1 shows photographs differentiated into vanA, vanB, and VanC types.
Resistance by vanA grows on the entire petri dish because of its high structural tolerance. In the case of VanB, tolerance is induced by vancomycin contained in the disc, so that a growth circle is observed only around the disc. In the case of VanC, tolerance is not induced by vancomycin contained in the disk, but a blocking circle is formed as a growth inhibitory action by vancomycin.
[0014]
Table 1 shows vanA-carrying vancomycin resistant E. coli. The result of faecium is shown.
Table 2 shows vanB-carrying vancomycin resistant E. coli. The result of faecium is shown.
Table 3 shows vanC owned E.I. The result of gallinarium is shown.
Table 4 shows vancomycin sensitivity. faecalis and E.M. The result of faecium is shown.
All strains with MIC values of 100 μg / ml or more for vancomycin and teicoplanin were confirmed by PCR with the vanA gene, which was 100% consistent with the results confirmed by this detection method. In addition, the vanB gene was confirmed by PCR for the vancomycin MIC value of 12.5 μg / ml or more and the teicoplanin MIC value of 1.56 μg / ml or less. Furthermore, vanComycin and teicoplanin-sensitive strains (MIC values of 1.56 μg / ml or less) were confirmed to have vanC gene by PCR, which was consistent with the results of this detection method.
From the above results, the results of the vanA, vanB, and vanC genes identified by the detection method of the present invention all coincided with the results of the vanA, vanB, and vanC genes confirmed by PCR.
[0015]
[Table 1]
[Table 2]
[Table 3]
[Table 4]
Example 2
<Comparison with commercially available vancomycin-resistant enterococci detection medium>
Comparison of detection results of commercially available vancomycin-resistant enterococci detection medium Enterococcosel medium (containing 8 μg / ml vancomycin) and Brain Heart Infusion Agar (containing 6 μg / ml vancomycin) (BHIA) was performed.
Table 5 shows vanA-carrying vancomycin resistant E. coli. The comparison result of the detection type with the commercially available medium of faecium is shown. Since resistance by vanA is constitutive high tolerance, it grows on the whole petri dish even if any medium is used.
[0020]
Table 6 shows a comparison result of detection type with vanB-carrying vancomycin-resistant E. faecium.
Since vancomycin contained in the disk induces resistance to vanB, a growth circle is observed only around the disk. Without induction of resistance by vancomycin, the growth is completely inhibited by teicoplanin contained in the medium.
Table 7 shows the comparison results of the detection type with vanC possessed E.gallinarium. Even vancomycin contained in the disk does not induce vanC resistance, but conversely forms a blocking circle as a growth inhibitory action by vancomycin.
Table 8 shows the comparison results of detection types with vancomycin sensitive E.faecalis and E.faecium. Vancomycin-sensitive bacteria do not grow in any medium.
[0021]
[Table 5]
[Table 6]
[Table 7]
[0024]
[Table 8]
【The invention's effect】
In the case of detecting vancomycin-resistant enterococci using a commercially available medium, vanA and vanB are observed to grow on the entire petri dish, and vanA and vanB cannot be differentiated. However, in the detection medium of the present invention, vanA grows on the entire surface, but vanB becomes a growth circle only around the disc and can distinguish between vanA and vanB. In addition, detection of vanC using a commercially available medium is partial growth from the entire surface (place where the amount of bacteria is deep), and in the case of full growth, there is no distinction between vanA and vanB. In addition, even in the case of partial growth, there is a case in which it is not possible to distinguish whether it is a false positive because the amount of bacteria is too thick or a positive because it has resistance. However, the detection method of the present invention can be clearly distinguished from vanA and vanB because it grows on the entire surface of the petri dish and a blocking circle is formed around the vancomycin-containing disc. Naturally, vancomycin-sensitive bacteria do not grow (not detected) using any detection medium.
[0026]
The vancomycin-resistant enterococci detection method of the present invention is a detection method that can be adequately handled by conventional testing techniques in clinical laboratories (bacteria laboratories), and it is not necessary to perform complicated PCR methods (vanA, vanB, VanC). Can be distinguished. Therefore, the examination time can be greatly shortened, and early treatment methods and therapeutic drugs can be selected. Furthermore, nosocomial infection of vancomycin-resistant enterococci has become an important problem, but if this detection method is used, not only can an early response be made to prevent nosocomial infections, but important information (vanA, vanB, differentiation of vanc gene) is also obtained.
[0027]
[Literature]
1) Leclercq R., E. Derlot, J. Duval, and P. Courvalin. Plasmid-mediated resistance to vancomycin and teicoplaninin in Enterococcus faecium. N. Engl. J. Med. 319: 157-161, 1988.
2) Courvalin P. Resistance of enterococci to glycopeptides. Antimicrob. Agent Chemother. 34: 2291-2296, 1991.
3) Arthur M., and P. Courvalin. Genetics and mechanisms of glycopeptide resistance in Enterococci. Antimicrob. Agents Chemother. 37: 1563-1571, 1993.
4) Clark NC, RCCooksey, BCHill, JMSwenson, and FCTenover.Characterization of glycopeptide-resistant Enterococci from UShospitals. Antimicrob. Agents Chemother. 37: 2311-2317, 1993.
[Brief description of the drawings]
FIG. 1 is a photograph showing differences in the growth of bacteria due to differences in vancomycin-resistant bacterial display genes.
Claims (5)
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