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1 Korean J Clin Microbiol Vol. 12, No. 2, June, 2009 Central Venous Catheter-Related Microbacterium Bacteremia Identified by 16S ribosomal RNA Gene Sequencing Chang-Jin Moon, Jong-Hee Shin, Eun-Sun Jeong, Seung-Jung Kee, Soo-Hyun Kim, Myung-Geun Shin, Soon-Pal Suh, Dong-Wook Ryang Department of Laboratory Medicine, Chonnam National University Medical School, Gwangju, Korea We describe here a case of central venous catheter (CVC)-related bacteremia caused by Microbacterium species in a 14-year-old patient, who had received chemotherapy for acute lymphoblastic leukemia. All nine blood cultures obtained from admission day 2 to day 62 yielded the same yellow-pigmented coryneform rod. Both Vitek 2 (biomerieux, USA) and MicroScan (Dade Behring, USA) identified the isolate as Micrococcus species, and the API Coryne (biomerieux, France) identified the isolate as Rhodococcus or Brevibacterium species. However, the 16S rrna gene sequence showed a 99% identity with Microbacterium species. The bacteremia was recurrent or persistent over 60 days despite alternate systemic antibiotic therapy, but blood culture became negative after an addition of teicoplanin lock therapy for eradicating CVC-related bacteremia. This represents the first report of CVC-related Microbacterium bacteremia cured by antibiotic lock therapy in Korea. (Korean J Clin Microbiol 2009;12:97-101) Key Words: 16S rrna sequencing, Catheter-related bacteremia, Microbacterium species 서 Microbacterium 균종은피부와인두에정상적으로상재하는그람양성막대균으로서, 임상검체에서분리되는노란색소를가진 coryneform 세균의대부분을차지한다 [1]. 최근 Microbacterium은임상검체에서분리되는빈도가증가하고있으나, 상대적으로감염을일으킬확률이낮아자주오염균으로간주된다 [2]. 이균종에의한감염은주로혈액종양또는항암화학요법등으로면역이저하된환자에서일어나며감염성심내막염혹은패혈증등이보고되고있다 [2-5]. 특히이균에의한혈류감염은중심정맥관사용과연관성이높으며전신성항생제의사용과동시에중심정맥관을제거함으로써회복됨이보고되었다 [2,3]. 국내에서사람의혈액에서 Microbacterium이분리된증례는현재까지두예가보고되었는데, 두예모두발열을주소로내원한심각한기저질환이없는환자였고, 그중 1예는감염성심내막염으로진단되었다 [4,6]. Microbacterium 균종은드물게인체감염을일으키고생화학적성상이다양하기때문에통상적방법으로동정하기가어렵 Received 16 February, 2009, Revised 6 April, 2009 Accepted 1 May, 2009 Correspondence: Jong-Hee Shin, Department of Laboratory Medicine, Chonnam National University Medical School, 671, Jebongno, Dong-gu, Gwangju , Korea. (Tel) , (Fax) , ( ) shinjh@chonnam.ac.kr 론 고, 정확한동정을위해서는대개 16S rrna 유전자염기서열비교가필요하다 [2,3]. 저자들은급성림프구성백혈병으로항암화학치료중인환자의혈액에서입원후 60여일동안 9차례연속분리된균을 16S rrna 유전자염기서열분석을이용하여 Microbacterium으로동정하였고, 이환자의중심정맥관연관균혈증을중심정맥관제거없이 antibiotic lock therapy로치료하였기에보고하는바이다. 증례 14세남자환자가 38.5 o C의발열, 복통, 구토및설사를주소로입원하였다. 환자는 2개월전급성림프구성백혈병으로진단받고관해유도치료시행및일차강화치료시행후회복중이었고백혈병진단이후혈관내중심정맥관 (dual lumen-cuffed catheter) 을유지하고있었다. 입원당시일반혈액검사소견은백혈구 400/mm 3 ( 중성구 73.6%), 혈색소 8.6 g/dl, 혈소판 42,000/mm 3 으로범혈구감소증을보이고있었다. 내원후실시한 9번의혈액배양 ( 내원 2일, 28일, 33일, 55일, 57 일, 58일, 59일, 61일및 62일 ) 에서모두동일한그람양성막대균이분리되었는데, 이균은혈액한천배지에서 48시간배양후연한황색의작은집락을형성하였다 (Fig. 1). 생화학적검사상 catalase 양성, oxidase 음성, 이동성 (motility) 음성이었으며 nitrate 환원검사음성, CAMP test 양성이었다. Vitek 2 (biomerieux, 97

98 Korean J Clin Microbiol 2009;12(2):97-101 Fig. 1.

(B) Yellow-pigmented colonies grown on blood agar plate after 48 hours of incubation at 37 o C. Fig. 2.

2 98 Korean J Clin Microbiol 2009;12(2): Fig. 1. (A) Gram staining of Microbacterium species showing irregular, short, thin gram positive rods (Gram stain, 1,000). (B) Yellow-pigmented colonies grown on blood agar plate after 48 hours of incubation at 37 o C. Fig. 2. Phylogenetic tree showing the relationship of the isolate from our patient to related species. Inc., Hazelwood, MO, USA) 동정결과는 Micrococcus luteus/ Micrococcus lylae였고, MicroScan WalkAway 96 system (Dade Behring, Sacramento, CA, USA) 동정결과는 Micrococcus species였으며 API Coryne system (biomerieux, Marcy-l'Etoile, France) 을이용한동정결과는 Rhodococcus species 혹은 Brevibacterium species이었다.

3 Chang-Jin Moon, et al. : Microbacterium Bacteremia 99 혈액배양에서자란균의 DNA를추출하여 16S rrna 유전자의 UFPL 및 URPL primer (5'-AGTTTGATCCTGGCTCAG-3', 5'-GGTTACCTTGTTACGACTT-3') 를이용하여 PCR을이용하여약 1,300 bp의염기서열을분석하였고 [7], 이를 Basic Local Alignment Search Tool (BLAST) database와 MEGA4 (megasoftware, Tempe, AZ, USA) 를이용하여 Neighbor-Joining method 로계통학적분류를시행하였다. 본증례의균주는 Microbacterium kitamiense (BLAST accession no. AJ717354) 및 Microbacterium chocolatum (BLAST accession no. AM18150) 과각각 99.0% 의일치율을보였다 (Fig. 2). Cefepime (3 g/day, 입원 2일부터 6일까지, 입원 24일부터 29 일까지, 입원 56일부터 59일까지 ) 과 ceftizoxime (3 g/day, 입원 13일부터 20일까지, 입원 46일부터 55일까지 ) 의정맥주사를교대로투여하여임상양상이일시적으로호전되었으나항생제치료를중단하면다시발열이발생하는현상이내원 60일까지반복되었다. 입원 55일째부터다시균혈증이발생하여지속적으로말초혈액배양에서동일균이분리됨에따라중심정맥관연관균혈증을의심하였고입원 61일째부터중심정맥도관을 teicoplanin으로채운후고농도로유지시키는 teicoplanin lock therapy를실시하였으며 ( 입원 61일부터 65일까지 ), 동시에 imipenem (2 g/day, 입원 61일부터 69일까지 ) 을정맥주사하였다. 입원 62일째중심정맥도관에서채취한혈액에서는동일한균이배양되었으나말초혈액배양에서는균이분리되지않았고, 이이후말초혈액이나중심정맥도관을통한혈액배양둘다에서균이더이상분리되지않았으며환자의발열증상도호전되었다. 환자는내원 71일째퇴원하였다. 고찰임상적으로드물게발견되는세균에의한감염은역학, 임상경과, 항생제감수성결과및생화학적성상등에대한정보가충분하지않기때문에일반임상미생물검사실에서이러한세균을정확히동정하기는어렵다. 일반적인검사로동정하기어려운세균을정확히동정하기위한여러분자유전학적검사법이개발되었고, 이중 16S rrna 유전자염기서열을비교하는방법이가장널리사용되고있다 [8-10]. 국내에서보고된 Microbacterium 증례 2예중한예는 16S rrna 유전자염기서열분석을통하여원인균을동정한반면 [2], 심내막염의예의경우원인균을 API Corynebacterium system (biomeriux, France) 으로동정하였다 [4]. 본증례에서는중심정맥관연관균혈증의원인균을 16S rrna 유전자염기서열분석을시행함으로써 Microbacterium 균속임을확인하였다. 일반적으로 Microbacterium의동정은대부분의임상미생물검사실에서일반적으로시행되는생화학적검사로는어렵다고알려져있다. 본균주는 Vitek 2와 MicroScan에의해 Micrococcus species로동정되었으며 API Coryne에의해서는 Rhodococcus species 혹은 Brevibacterium species로동정되었다. 그람염색상 Micrococcus species는그람양성의알균이사량체 (tetramer) 를이루는것이특징인데비해, 본균은그람양성의막대균으로서감별할수있었다. 또한본균은 Rhodococcus species 혹은 Brevibacterium species와는달리배양검사에서황색의작은집락을형성하여서쉽게감별이가능하였다. 본증례에서동정된 Microbacterium 균주는 Laffineur 등 [3] 이대부분의 Microbacterium 균종에서반응을보이지않는다고보고한 glycerol 및 erythritol에대해서반응을보이지않아, 균속이일치하는소견을보였다. 한편, 임상검체에서분리되는노란색소를가진 coryneform 세균으로는 Microbacterium 이외에 Cellulomonas 균종이있다. Cellulomonas는 urea 음성, casein 가수분해음성, nitrate 양성이고 sucrose로부터산을생성하는반면 Microbacterium은이 4가지검사에모두에대해다양한반응을보일수있으나확실한동정을위해서는 16S rrna 유전자염기서열분석이요구된다 [2]. 따라서본증례는통상적상품화된제품을이용하여본균속을동정할경우다른균종으로잘못동정될가능성을보여주었다. CLSI (the Clinical and Laboratory Standards Institute) MM18-A 지침에따르면 16S rrna 유전자염기서열분석에서 coryneform 그람양성막대균의경우 97% 이상일치하면동일균속으로동정할수있고, 99% 이상일치하면동일균종으로동정할수있다 [11]. 그런데, 본균주는 16S rrna 유전자염기서열분석을실시하여 BLAST database 를이용하여계통학적분류를시행한결과, M. kitamiense 및 M. chocolatum (BLAST accession no. AM18150) 의두균종와각각 99% 의높은일치율을보였다. Takeuchi와 Hatano에따르면 [12], M. chocolatum의경우 D-arabinose 음성, D-mannose 양성, D-mannitol 양성, N-acetyl glucosamine 음성, D-maltose 양성등의특징을보이는데이는본증례의결과와모두일치하였다. 그러나 M. kitamiense의경우현재보고가 1예밖에없고 [13], 이보고에서는상세한생화학적양상을밝히지않아서본균주와비교가어려웠다. 따라서이두균종과다른균종을감별할수있는생화학및다른유전자의염기서열분석양상에대한추가정보가필요한데, 현재이에대한보고가거의없어 Microbacterium 균종내의다른균종과감별동정은어려울것으로생각되었다. Lau 등 [2] 은 16S rrna 유전자부위가서로다른 Microbacterium 균종사이에매우보존적이어서 (highly conserved), 균종을구분할수있다기보다는 Microbacterium의균속수준 (genus level) 까지만동정이가능하다고하였는데, 본증례에서도 Microbacterium 균종의동정에있어유전자염기서열분석법의한계성을확인할수있었다 [2]. 임상적으로중요한 Microbacterium 균종에의한감염보고는현재세계적으로약 20예가보고되고있으며주로감염성심내

4 100 Korean J Clin Microbiol 2009;12(2): 막염, 내안구염및균혈증등이고최근이균에의한균혈증의집단발병도보고되었다 [2-5]. 국외에서혈액종양환자에서항암화학요법중인환자에서 Microbacterium 균종에의한중심정맥관연관균혈증이현재까지약 6예정도보고되고있는데 [2,3], 국내에서는면역저하환자에서의중심정맥관연관감염은보고된바없다. 중심정맥관관련패혈증의진단에는카테터를제거하여반정량적으로 tip을배양하는방법이주로이용되고있으나 [14], 중심정맥관을제거하지않고유지하면서중심정맥관관련패혈증을진단할수있는정량혈액배양법, 중심정맥관을통한혈액과말초혈액의배양에서의양성검출시간차를이용하는법등도이용되고있다 [15]. 본환자의경우중심정맥관제거후 tip 배양이나정량혈액배양법등이의뢰되지않아이방법을이용한중심정맥관연관균혈증진단은하지못하였으나입원 62일에중심정맥도관을통한혈액과말초혈액에서동시에혈액배양을실시한결과, 중심정맥도관을통한혈액에서는균이배양되었으나말초혈액배양에서는균이분리되지않은점과중심정맥관에 antibiotic lock therapy로균혈증이회복된점으로중심정맥관연관균혈증으로진단할수있었다. 따라서본증례는급성림프구성백혈병환자에서발생한 Microbacterium 균종에의한중심정맥관연관균혈증의국내첫증례로생각된다. Alonso-Echanove 등이 2001년에보고한바에의하면면역저하환자에있어서 Microbacterium 균종의감염은높은유병률과사망률을나타내었다 [5]. 국외에서보고된중심정맥관연관균혈증의경우대개 vancomycin, penicillin G 및 ampicillin 등항균제를전신적으로투여하고중심정맥도관을제거하여치료하였는데 [2,3], 본증례의경우지속적인항암화학치료를위해중심정맥도관을제거할수없는상황이었다. 환자는 3세대및 4세대 cephalosporin 치료에일시적인임상양상의호전을보이고혈액배양에도음성을보였지만항생제투여를중단하면발열이재발하고다시혈액배양에서도동일균이분리되었다. 따라서중심정맥관연관균혈증을의심하고혈관내유치도관연관감염증의치료지침에따라 teicoplanin 을중심정맥도관에채우고고농도로유지하였다 [16]. 최근중심정맥관을교환하지않고도관내의공간에항생제를채워넣는 antibiotic lock 치료가중심정맥관연관균혈증을효과적으로치료할수있다는보고들이있으나이에의한치료율은원인균주에따라서다르게나타난다. 예를들면 Staphylocoocus aureus 보다 coagulase 음성 staphylococci에의한감염이더좋은치료성적을나타낸다 [17]. 본증례는 Microbacterium 균속에의한중심정맥관연관균혈증을 antibiotic lock 치료에의해치료될수있음을처음으로보여준예라생각된다. 결론적으로본증례는입원환자, 면역저하환자및중심정맥관을사용중인환자에서노란색소를가진 coryneform 세균이분리될경우 Microbacterium 균종의가능성을고려해야하며 Microbacterium의동정은상품화된제품만으로는정확히동정하지못할가능성이있으므로 16S rrna 유전자염기서열분석을고려해야함을보여주었다. 참고문헌 1. Funke G and Bernard KA. Coryneform Gram-Positive Rods. In: Murray PR, Baron EJ, et al. eds. Manual of Clinical Microbiology. 9th ed, Washington; ASM Press, 2007: Lau SK, Woo PC, Woo GK, Yuen KY. Catheter-related Microbacterium bacteremia identified by 16S rrna gene sequencing. J Clin Microbiol 2002;40; Laffineur K, Avesani V, Cornu G, Charlier J, Janssens M, Wauters G, et al. Bacteremia due to a novel Microbacterium species in a patient with leukemia and description of Microbacterium paraoxydans sp. nov. J Clin Microbiol 2003;41: Kim CH, Suk JE, Han WS, Kim YH, Hwang BY, Jeong HW, et al. A case of native valve infective endocarditis caused by Microbacterium species. Korean J Med 2004;67(Supple):S923-S6. 5. Alonso-Echanove J, Shah SS, Valenti AJ, Dirrigl SN, Carson LA, Arduino MJ, et al. Nosocomial outbreak of Microbacterium species bacteremia among cancer patients. J Infect Dis 2001;184: Ko KS, Oh WS, Lee MY, Peck KR, Lee NY, Song JH. A new Microbacterium species isolated from the blood of a patient with fever: Microbacterium pyrexiae sp. nov. Diagn Microbiol Infect Dis 2007;57: Lipuma JJ, Dulaney BJ, McMenamin JD, Whitby PW, Stull TL, Coenye T, et al. Development of rrna-based PCR assays for identification of Burkholderia cepacia complex isolates recovered from cystic fibrosis patients. J Clin Microbiol 1999;37: Fredricks DN and Relman DA. Sequence-based identification of microbial pathogens: a reconsideration of Koch s postulates. Clin Microbiol Rev 1996;9: Harmsen D, Rothganger J, Frosch M, Albert J. RIDOM: Ribosomal differentiation of medical micro-organisms database. Nuclei Acids Res 2002;30: Petti CA. Detection and identification of microorganisms by gene amplification and sequencing. Clin Infect Dis 2007;44: Clinical and Laboratory Standards Institute. Interpretive criteria for identification of bacteria and fungi by DNA target sequencing; approved guideline. CLSI document MM18-A. Wayne, PA; CLSI, Takeuchi M and Hatano K. Proposal of six new species in the genus Microbacterium and transfer of Flavobacterium marinotypicum ZoBell and Upham to the genus Microbacterium as Microbacterium maritypicum comb. nov. Int J Syst Bacteriol 1998;48: Matsuyama H, Kawasaki K, Yumoto I, Shida O. Microbacterium kitamiense sp. nov., a new polysaccharide-producing bacterium isolated from the wastewater of a sugar-beet factory. Int J Syst Bacteriol 1999;49: Maki DG, Weise CE, Sarafin HW. A semiquantitative culture method for identifying intravenous-catheter-related infection. N Engl J Med 1977;296: Blot F, Schmidt E, Nitenberg G, Tancre de C, Leclercq B, Laplanche A, et al. Earlier positivity of central-venous- versus peripheralblood cultures is highly predictive of catheter-related sepsis. J Clin Microbiol 1998;36: Mermel LA, Farr BM, Sherertz RJ, Raad II, O Grady N, Harris JS,

5 Chang-Jin Moon, et al. : Microbacterium Bacteremia 101 et al. Guidelines for the management of intravascular catheterrelated infections. Clin Infect Dis 2001;32: Poole CV, Carlton D, Bimbo L, Allon M. Treatment of catheter- related bacteremia with an antibiotic lock protocol: effect of bacterial pathogen. Nephrol Dial Transplant 2004;19: = 국문초록 = Microbacterium 에의한중심정맥관연관균혈증 1 예 : 16S rrna 유전자염기서열분석을이용한균동정 전남대학교의과대학진단검사의학교실문창진, 신종희, 정은선, 기승정, 김수현, 신명근, 서순팔, 양동욱 저자들은급성림프구성백혈병으로진단받고, 관해유도치료및일차강화치료시행후회복중이던 14세남아에서 Microbacterium에의한중심정맥관연관균혈증을경험하였기에보고하는바이다. 내원후 2일부터 62일까지시행한 9차례의혈액배양에서황색의작은집락을형성하는그람양성막대균이지속적으로분리되었다. 이균은 Vitek 2 (biomerieux, USA) 와 MicroScan WalkAway 96 system (Dade Behring, USA) 에의해 Micrococcus spp. 로, API Coryne system (biomerieux, France) 에의해 Rhodococcus spp. 및 Brevibacterium spp. 로동정된반면, 16S rrna 유전자염기서열분석결과, Microbacterium 균종과 99% 의일치율을보였다. 전신적인항생제치료에도불구하고환아의균혈증은 2개월간지속되었으나, 중심정맥관연관균혈증치료를위한 teicoplanin lock therapy 시행후혈액배양에서더이상균이자라지않았다. 본증례는국내에서중심정맥관연관 Microbacterium 균혈증에관한첫증례보고이다. [ 대한임상미생물학회지 2009; 12:97-101] 교신저자 : 신종희, , 광주시동구제봉로 671 전남대학교병원진단검사의학과 Tel: , Fax: shinjh@chonnam.ac.kr

( )Kju225.hwp

( )Kju225.hwp 비임균성비뇨생식기감염에서 의진단적효용성 Usefulness of the Mycofast Test (MYCOFAST Evolution 2) for the Diagnosis of Nongonococcal Genitourinary Infections Hang Ro Park, Yang Hyun Kim, Ho Jae Lee, Jea Sang Oh, Hyoung Jin