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16 Byoung-Seon Yang, et al. Rapid Detection of VRE ORIGINAL ARTICLE Korean J Clin Lab Sci. 2013, 45(1):16-20 ISSN 1738-3544 Rapid Detection of Vancomycin-resistant Enterococci (VRE) in Clinical Samples from University Hospital Byoung-Seon Yang 1, Jung-Yeon Park 1, and Seung-Gu Choi 2 1 Department of Clinical Pathology, Jinju Health College, Jinju 660-757, Korea 2 Department of Clinical Laboratory Science, Shinheung College, Uijeongbu 480-701, Korea Outbreaks of vancomycin-resistant enterococci (VRE) are being reported more frequently in many countries. While seven glycopeptide resistance genotypes have been described in Enterococci, vana and vanb are the most common resistance genotypes. The aim of this study was to detect antibiotic susceptibilities of 23 Enterococcus faecium strains, which caused an outbreak in a University hospital by a disk diffusion test to investigate the presence of the species specific gene, and the resistant genotypes, vana and vanb by duplex PCR. PCR for vana and vanb was performed on 23 enterococci. Twenty three were identified as E. faecium and were tested positive for the vana genotype. This study will report on the validation of a simple and accurate VRE detection method that can be easily incorporated into the daily routine of a clinical laboratory. Early detection of VRE strains, including those with susceptibility to vancomycin, is of paramount clinical importance as it allows rapid initiation of strict infection control practices, as well as the therapeutic guidance for confirmed infections. The PCR method developed in the present study is simple and reliable for the rapid characterization of VRE. Keywords: Vancomycin-resistant enterococci (VRE), vana, vanb Corresponding author: Byoung Seon Yang Department of Clinical Pathology, Jinju Health College, Jinju 660-757, Korea. Tel: 82-55-740-1851 E-mail: ybseon@hanmail.net This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright 2013 Korean Society of Clinical Laboratory Science. All rights reserved. Received: January 27, 2013 Revised: February 7, 2013 Accepted: March 13, 2013 서론 Enterococci 는건강한성인의경우대장에상주하는정상상재균이다 (Elsner 등, 2000). 현재심내막염, 균혈증, 비뇨기계감염과신생아패혈증등의환자에있어서병원내감염으로증가추세에있다 (Dutka-Malen 등, 1995). Enterococcus faecalis와 E. faecium은임상에서가장많이분리되는종이며, E. avium, E. durans, E. casseliflavus, E. gallinarum과 E. flavescens 균주는 5% 이하로드물게분리된다 (Papaparaskevas 등, 2002). 1980년대이후 vancomycin-resistant enterococci (VRE) 는원내감염을일으키는중요한세균그룹으로알려졌다. 병원에서얻은균혈증의대부분을차지한다 (Cheng 등, 1997). VRE는다양한항생제에저항성을나타내고, 5종의내성유전자는 vana, vanb, vand, vane 그리고 vang로획득유전자이다. VanC1, vanc2/c3 2종의유전자는내인성유전자이다. VanA와 vanb 유전자를가진 VRE가임상에서가장많이분리된다 (Perez-Hernamdez 등, 2002). VRE유전자 는접합성 plasmid나 transposon 에위치한다 (Radu 등, 2001). vana 유전자를가진 Enterococci 는 vancomycin과 teicoplanin 의고농도에저항성을보이고 vanb의경우는 vancomycin만내성을보인다. vanc의경우는 vancomycin 의저농도에내성을보인다 (Bell 등, 1998). VRE 전파는균주자체가이동하는수직전이와내성유전자만이전파되는수평전이기전에의하며, VRE 전파를방지하기위하여대부분의대학병원에서는 centers for disease control and prevention (CDC) 의 hospital infection control practices advisory committee (HICPAC) 기준에따르고있다. VRE 집락이형성된환자는신속히격리하고적어도 1주이상의간격으로시행한감시배양에서연속적으로 3회음성인경우격리를해지한다. VRE의정확한신속검출은병원내감염전파의추적과관리를위해서필요하고, 또한 VRE가분리되는환자에있어서장내집락화의유무를신속하게검출하여사람간의전파를방지해야한다 (Devriese 등, 1993). VRE을관리하기위해서신속하고정확한검

Korean J Clin Lab Sci. Vol. 45, No. 1, Mar. 2013 17 출이필요하다. 배양에기초한표현형에의한 VRE 검출은시간이많이소요되고, 저농도 glycopeptide 내성세균검출이어렵다 (Elsner 등, 2000). PCR방법은임상분리균주의검출및항생제내성유전자분석에특이적이고, 시간적인소모가적고분석력과재현성이좋은방법이다 (Monstein 등, 1998). 본연구에서는대학병원으로부터 VRE균주를분리하여종특이유전자및 vana, vanb 유전자를대상으로 duplex PCR 방법을실시하여표현형과유전자형을비교분석하였다. 재료및방법 1. 재료 2012년 9월부터 11월까지대전 C 대학병원에서 VITEK (bio- Merieux, Inc. Haxelwood, MO, USA) 기기로 vancomycinresistance Enterococci 세균으로동정된 23균주를대상으로하였다. 분리균주동정및항생제감수성검사정도관리균주로 Enterococcus faecium ATCC 19434, Enterococcus casseliflavus ATCC 700327 균주를사용하였다. 2. Enterococcus종동정세균부유액을혈액한천배지에접종한후 30 o C 배양기에서 18 24시간배양후집락및용혈유무를관찰하였다. 순수분리집락을도말하여그람염색을실시하였다. 생화학적시험으로 catalase test, bile esculin test, 6.5% NaCl 배지에서성장유무를관찰하였다. 3. 항생제감수성시험세균부유액을 Mueller-Hinton Agar (MHA) 에고르게접종한후, 배지의중앙에는 vancomycin (30 μg) 디스크와 teicoplanin (30 μg) 있는디스크를 20 mm 간격으로놓았다. 접종배지는 37 o C 항온기에 18시간배양후결과를판정하였고 minimal inhibitory concentration (MIC) 는 MH액체배지에 vancomycin 항생제는 400 μg/ml, 350 μg/ml, 300 μg/ml, 250 μg/ml, 200 μg/ml로 희석, teicoplanin 항생제는 128 μg/ml, 64 μg/ml, 32 μg/ml, 16 μg/ml, 8 μg/ml희석하여균주접종후배양하여판독하였다. 4. 균주로부터의 DNA 분리 23개의 VRE 균주를가온법을이용하여 DNA를추출하였다. 순수분리된 23개균주를 90 o C에서 10분방치후진탕하였다. 그리고 13,000 rpm에서 5분간원심후상층액을얻었다. 5. Duplex PCR 을위한 primer 의합성 Enterococcus 종동정및 VRE 유전자를분석하기위한 primer 를아래과같이제작하였다 (Table 1). 6. Duplex PCR 을이용한 Enterococcus 종및 van 유전자의증폭 Duplex PCR을위한 PCR 혼합액 dntp ( 각기 2.5 mm), 10 PCR buffer 2 μl, primer 2쌍 10 pmol 각각 1 μl씩넣었고, genomic DNA (25 μg) 1 μl, Taq DNA polymerase (2 unit) 1 μl, 최종반응액은증류수로 20 μl 되게끔실시하였다. DNA thermal cycler (Perkin Elmer, Inc. Wellesley, MA, USA) 에서 Enterococcus 종동정은 94 o C에서 2분간변성, 58 o C에서 1분간접합, 68 o C에서 1분간확장의순서로 30회를시행하고 68 o C에서 7분간최종확장하였다. Van 유전자검출은 95 o C에서 1분간변성, 64 o C에서 2분간접합, 72 o C에서 1분 30초간확장의순서로 35회를시행하고 72 o C 에서 7분간최종확장하였다. Table 1. PCR primers used in this study Primer Enterococcus spp. (ddl gene) Ent1-F Ent2-R Van gene Van AB-F Van A-R Van B-R Sequence GCA AGG CTT CTT AGA GA CAT CGT GTA AGC TAA CTT C GTA GGC TGC GAT ATT CAA AGC CGA TTC AAT TGC GTA GTC CAA GCC GAC AAT CAA ATC ATC CTC Product length (bp) 587 231 330 Fig. 1. Glycopeptide susceptibility of VRE as tested by disk diffusion test. VA, vancomycin (30 μg/ml); TEC, teicoplanin (30 μg/ml).

18 Byoung-Seon Yang, et al. Rapid Detection of VRE 7. PCR 산물의확인 PCR 증폭산물을 1.5% agarose gel에 midori green (2 μl) 을넣어 1 TAE buffer에서 70 volt, 100 ma로 45분동안전기영동을실시한다음, agarose gel을 UV transilluminator로관찰하고, Gel Doc (Bio-Red, Inc. Hercules, CA, USA) 이미지분석장치로사진촬영하였다. 결과 1. Enterococcus종동정혈액한천배지에서다양한용혈양상을보이고, 그람염색결과그람양성쌍알균, 사슬알균형태로나타났다. 생화학적시험결과 catalase음성, bile esculin양성, 6.5%NaCl 에서성장하였다. 2. 항생제감수성시험 디스크감수성시험에서는 vancomycin 디스크에서는 1 23 균 Table 2. Distribution of 23 strains of vancomycin-resistant Enterococci from university hospital isolates, susceptibility profile and genotype Strains DDT Phenotype MIC Genotype Van Tec Van Tec VanA VanB Enterococcus faecium 1 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 2 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 3 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 4 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 5 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 6 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 7 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 8 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 9 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 10 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 11 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 12 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 13 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 14 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 15 R R 250 μg/ml 32 μg/ml - - Enterococcus faecium 16 R R 250 μg/ml 32 μg/ml + - Enterococcus faecium 17 R R 320 μg/ml 64 μg/ml + - Enterococcus faecium 18 R R 320 μg/ml 64 μg/ml + - Enterococcus faecium 19 R R 320 μg/ml 64 μg/ml + - Enterococcus faecium 20 R R 320 μg/ml 64 μg/ml + - Enterococcus faecium 21 R R 320 μg/ml 64 μg/ml + - Enterococcus faecium 22 R R 320 μg/ml 64 μg/ml + - Enterococcus faecium 23 R R 320 μg/ml 64 μg/ml + - DDT, disk diffusion test; MIC, minimum inhibition concentration; Van, vancomycin; Tec, teicoplanin; R, resistance. Fig. 2. Electrophoresis of the amplified products of Enterococcus spp. By a duplex PCR in a 1.5% agarose gel. Lane M, 100 bp DNA ladder; lanes 1 to 14 and 16 to 23, E. faecium; lane N, negative control.

Korean J Clin Lab Sci. Vol. 45, No. 1, Mar. 2013 19 Fig. 3. Electrophoresis of the amplified products of VanA (231 bp), VanB (330 bp) genes. By a duplex PCR in a 1.5% agarose gel. Lane M, 100 bp DNA ladder; lanes 1 to 14 and 16 to 23, VanA; lane N, negative control. 주 (100%) 가내성으로나타났으며, 또한 teicoplanin 디스크에모든균주가내성으로나타났다 (Fig. 1). MIC 결과 vancomycin 항생제의경우 1 16 균주가 250 μg/ml, 17 23 균주가 320 μg/ml에서억제가되었으며 teichoplanin 항생제의경우 1 16 균주가 32 μg/ml, 17 23 균주가 64 μg/ml에서억제가되었다 (Table 2). 3. Enterococcus종동정및 VRE유전형 Duplex PCR에서 22균주 (95%) 가 E. faecium으로동정되었으며, 15번균주는검출되지않았다 (Fig. 2). VanA 생성 Enterococcus 균주는 22균주 (95%) 가양성으로나타났으며 15번균주가음성으로나타났으며, VanB생성 Enterococcus균주는검출되지않았다 (Fig. 3). 고찰 Enterococci 는사람뿐만아니라동물, 식품으로부터분리된다. 항생제내성인 vancomycin-resistant enterococci (VRE) 균주가증가추세에있다. VRE균주는병원내감염을일으키고중환자실이나혈액종양병동등에서토착화되어가고있는실정이다. VRE균주의유행과전파를막기위한감염관리지침이있으나모든병원에서적용하기가어려운실정이고다각적감염관리를시행하여도근절하기힘든병원감염의주요다제내성균이다 (Facklam 등, 1989). 기존배양법을이용한경우검출까지의시간이최소한 2 3 일걸리므로검사소요시간을줄이기위해서대변이나직장도말검체에서직접 DNA를추출하여 Enterococcus종동정및 VRE내성유전자를검출하는방법들에관한보고가있으나검체에서직접 DNA을추출하는경우검체에포함된억제물질로인해중합효소연쇄반응을저해하는경우가있다. 전통적인방법으로 Enterococcus종동정하는경우 Lancefield group D 항원검사, 6.5% NaCl성장유무, pyrolidonydonylarylamidase시험, leucinearylamidase test와 bile esculin test를사용하여 Enterococcus를동정한다. 순수분리 배양해서종동정까지 24시간에서 48시간걸린다. 자동화동정시스템을사용하더라도배양에서종동정까지 24시간이상걸린다. 종동정이되면항생제감수성검사를실시하는데 vancomycin 내성유전자는 Enterococcus종사이에전이가가능하다. 또한 VRE검출이지연되면 VRE균주가집락화되어병원감염의문제가된다. 현재 DNA를기초한직접검출방법을많이사용하고있다. E. faecalisspecific과 E. faecium-specific PCR검출방법은다양한유전자를표적으로한다. dd l (D-alanine-D-alanine ligase coding) 유전자는 E. faecalis와 E. faecium종을특이적으로검출하고 PBP5 (penicillin binding protein coding) 유전자는 E. faecalis종을특이적으로검출한다. vanc 유전자는 E. gallinarum, E. casseliflavus 와 E. flavescens을검출한다 (Duka-Malen 등, 1995). 본실험에서표현형적방법으로분리된 VRE 23균주를디스크감수성시험과 MIC 로확인결과모든균주가내성으로나타났다. Duplex PCR에서 22균주 (95%) 가 E. faecium으로동정되었으며, E. faecium 15 균주는검출되지않았다. VanA 생성 Enterococcus균주는 22균주 (95%) 가양성으로나타났으며 E. faecium 15균주가음성으로나타났으며, VanB 생성 Enterococcus균주는검출되지않았다. VanA 생성 E. faecium에의한병원내집단발병시직장도말검체를액체배지에서증균시킨후 vana에특이한 PCR 로확인하는방법은 24 30시간내에 95% 의민감도와 100% 의특이도를보여평균 4 5 일이걸리는배양법의민감도및특이도와비교할때우수하다고하였다 (Roger 등, 1999). 또한 Kim 등 (2007) 도내부대조물질을사용하여위음성을확인하고, 선택액체배지를이용한신속 PCR을시행하는것이 VRE 보균여부를감시하는데빠르고민감한방법이라고하였지만 vana 이외에다른유전자형을검출하려면다중PCR을시행해야하며민감도가떨어질가능성이있다고하였다. 본실험에서 E. faecium 15균주의내성유전자형이검출되지않은것은유전자의변이가있을것으로예상된다. 대부분의 Enterococcus 균주에서는 vana 유전자를포함하고있는것으로나타났으며이방법은 vana, vanb 유전자의검출과이유전자의 monitoring에중요한역할을할것이라생각된다. 따

20 Byoung-Seon Yang, et al. Rapid Detection of VRE 라서분자생물학적기법인 duplex PCR을이용한 VRE의검출은표현형적방법에서의문제점의해결이나, 원내감염의역학에아주유용한방법이라사료된다. 참고문헌 Bell JM, Paton JC, Turnidge J. Emergence of vancomycin-resistant enterococci in Australia: phenotypic and genotypic characteristics of isolates. J Clin Microbiol. 1998, 36:2187-2190. Cheng S, McCleskey FK, Lin M, Gress HJ, Petroziello JM, Lin R, et al. A PCR assay for identification of Enterococcus faecium. J Clin Microbiol. 1997, 35:1248-1250. Devriese LA, Pot B, Collins MD. Phenotypic identification of the genus Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups. J Bacteriol. 1993, 75: 399-408. Dutka-Malen S, Evers S, Courvalin P. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol. 1995, 33: 24-27. Elsner HA, Sobottka I, Feucht HH, Harps E, Haun C, Mack D, et al. Nosocomial outbreak of vancomycin-resistant Enterococcus faecium at a German university pediatric hospital. Int J Hyg Envir Heal. 2000, 203:147-152. Facklam RR, Collins MD. Identification of Enterococcus species isolated from human infections by a conventional test scheme. J Ind Microbiol. 1989, 27:731-734. Kim S. Sung H, Jeon HS, Park SJ, Park SH, Kim MN. Evaluation of a rapid enrichment-pcr method for the detection of vana vancomycin-resistant enterococci in fecal specimens. Korean J Clin Microbiol. 2007, 10:44-8. Monstein HJ, Quednau M, Samuelsson A, Ahrne S, Isaksson B, Jonasson J. Division of the genus Enterococcus into species groups using PCR-based molecular typing methods. Microbiology. 1998, 144:1171-1179. Papaparaskevas J, Tassios PT, Kalapothaki V, Avlami A, Legakis NJ, Vatopoulos AC. Epidemiology of multiresistant Enterococcus avium isolates in a Greek tertiary care hospital. Int J Antimicrob Agents. 2002, 20:432-437. Perez-Hernandez X, Mendez-Alvarez S, Claverie-Mertin F. A PCR assay for rapid detection of vancomycin-resistant enterococci. Diagn Microbiol Infect Dis. 2002, 42:273-277. Radu S, Toosa H, Rahim RA, Reezal A, Ahmad M, Hamid AN, et al. Occurrence of the vana and vanc2/c3 genes in Enterococcus species isolated from poultry sources in Malaysia. Diagn Microbiol Infect Dis. 2001, 39:145-153. Roger M, Faucher MC, Forest P, St-Antoine P, Couttlee F. Evaluation of vana-specific PCR assay for detection of vancomycin-resistant Enterococcus faecium during a hospital outbreak. J Clin Micorbiol. 1999, 37:3348-3349.