증례 Lab Med Online Vol. 2, No. 2: , April 임상미생물학 재택자가항생제치료중이던환아에서발생한중심정맥관관련 Tsukamurella inchone

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1 증례 Lab Med Online Vol. 2, No. 2: , April 2012 임상미생물학 재택자가항생제치료중이던환아에서발생한중심정맥관관련 Tsukamurella inchonensis 균혈증 1 예 A Case of Catheter-Related Bloodstream Infection by Tsukamurella inchonensis in a Pediatric Patient Receiving Home Intravenous Antibiotic Treatment 서유경 1 정혜선 1 이양순 1 김주원 1 용동은 1 정석훈 1 한석주 2 이경원 1 Youkyung Seo, M.D. 1, Hae-Sun Chung, M.D. 1, Yangsoon Lee, M.D. 1, Juwon Kim, M.D. 1, Dongeun Yong, M.D. 1, Seok Hoon Jeong, M.D. 1, Seok Joo Han, M.D. 2, Kyungwon Lee, M.D. 1 연세대학교의과대학진단검사의학교실 1, 외과학교실 2 Department of Laboratory Medicine 1 and Research Institute of Bacterial Resistance, Department of General Surgery 2, Yonsei University College of Medicine, Seoul, Korea Bacteria belonging to the genus Tsukamurella are aerobic, gram-positive rods that are weakly acid-fast with no apparent branching. Infections of the Tsukamurella spp. are generally caused by the use of infected medical devices such as central venous catheters. The underreporting of these infections might be attributable to the frequent misdiagnosis of Tsukamurella infections as Corynebacterium or atypical Mycobacterium spp. infections. Therefore, when gram-positive aerobic rods are observed in the blood culture of a patient with a central venous catheter, it is important to consider Tsukamurella as one of the causative organisms. Here, we report the first case of a catheter-related blood stream infection caused by Tsukamurella inchonensis in a 3-yr-old Korean girl with underlying biliary atresia who underwent hepatoportoenterostomy. Key Words: Catheter, Blood stream infection, Tsukamurella inchonensis, 16S ribosomal RNA 서론 Tsukamurella 속은 1971 년 Tsukamura 등이 Corynebacterium paurometabolum 을 Tsukamurella paurometabola 로보고한이 래현재까지 11 개의균종이보고되었다. 이들균속은호기성방선 균 (actinomycetes) 중 Corynebacteriaceae 과에속하며세포벽에 mycolic acid 외에 meso-diaminopimelic acid 와 arabinogalactan 을 가지고, 약한항산성을띠며운동성이없고, 아포와균사를형성하 Corresponding author: Dongeun Yong, M.D. Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemu-gu, Seoul , Korea Tel: , Fax: , deyong@yuhs.ac Received: August 2, 2011 Revision received: October 11, 2011 Accepted: November 15, 2011 This article is available from , Laboratory Medicine Online This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 지않는그람양성막대균이다 [1]. Tsukamurella 속에의한인체감염은호흡기감염 [2, 3], 인공관절감염 [4], 복막염 [5], 결막염 [6], 각막염 [7] 등이보고되어있고, 이균속에의한혈류감염은정맥관과흔히연관되어있다 [8-10]. Tsukamurella 속은 16S rrna 염기서열상동성이 99% 이상으로높고, 생화학적성상또한비정형적인경우가많아흔히 Corynebacterium 이나비정형 Mycobacterium 으로잘못동정되기가쉽다 [11, 12]. 국내에서는 Tsukamurella pulmonis에의한정맥관관련혈류감염 [8, 12] 과 Tsukamurella tyrosinosolvens 에의한복막염 [13] 증례보고가있었다. 저자들은담도폐쇄증으로간문-장문합술 (hepatoportoenterostomy) 수술후발생한 Tsukamurella inchonensis 에의한패혈증을보고하고자한다. 증례 3년 6개월된환아가 2일간반복되는 38.2 의발열을주소로응급실을경유하여소아외과에입원하였다. 환아는담도폐쇄증으로출생 8주째간문-장문합술수술후가정에서중심정맥관으로 cefpiramide 0.16 mg을하루세번투여하던중임의로항생제투여를 eissn

서유경 외: T. inchonensis Bloodstream Infection 중단하고 7일만에 고열(38.2 )이 발생하였다.

4 mg/dl, 감마-글루타밀 트랜스펩티다제(gamma-glutamyl 69.9%), C 반응성 단백(C-reactive protein, CRP) 0.

2 로 발열이 있었고, 알칼리성인산가수분해 입부위의 발적이나 부종과 같은 염증소견은 관찰되지 않았다.

aminotransferase, AST) 75 IU/L, 알라닌 아 염을 의심하였다. 경험적으로 cefoperazone-sulbactam, amikacin, A B C D Fig. 1.

2 서유경 외: T. inchonensis Bloodstream Infection 중단하고 7일만에 고열(38.2 )이 발생하였다. 입원 당시 중심정맥 미노트란스페라제(alanin aminotransferase, ALT) 66 IU/L, 총빌리 관을 유지한 상태이었고 말초혈액 백혈구수 5,780/μL (분엽호중구 루빈 1.4 mg/dl, 감마-글루타밀 트랜스펩티다제(gamma-glutamyl 69.9%), C 반응성 단백(C-reactive protein, CRP) mg/dl로 정 transpeptidase, γ-gt) 238 IU/L로 증가되어 있었다[14]. 카테터 삽 상이었으나, 체온 38.2 로 발열이 있었고, 알칼리성인산가수분해 입부위의 발적이나 부종과 같은 염증소견은 관찰되지 않았다. 발 효소(alkaline phosphatase, ALP) 4,971 IU/L, 아스파탐 아미노트 열과 빌리루빈 증가, 간문-장문합술수술 과거력을 고려하여 담낭 란스페라제(aspartate aminotransferase, AST) 75 IU/L, 알라닌 아 염을 의심하였다. 경험적으로 cefoperazone-sulbactam, amikacin, A B C D Fig. 1. Macroscopic and microscopic features of the isolated strain. (A) Growth of the strain after 72 hr on blood agar plates; (B) A colony of the strain after 72-hr growth ( 10); (C) Gram stain of the strain after a 48-hr growth ( 1,000); (D) Modified acid-fast bacilli (AFB) stain of the strain after a 48-hr growth ( 1,000) 106

3 metronidazole 을투여하였다가, 혈액배양에서그람양성막대균이검출됨을확인한이후에는 cefpiramide, panipenem, metronidazole을동시에투여하였고, 입원 4일째발열증상이소실되어다시항생제를재택자가투여할것을계획하고입원 9일후퇴원하였다. 입원당시말초정맥혈액을 2쌍의호기성배지와혐기성배지에접종하여 48시간배양한후 2쌍중 2병의호기성배지에서그람양성막대균 (YMC11-5-B1957) 이관찰되었다. 혈액배양액을혈액한천배지에접종하여 35, 5% CO 2 에서 72시간배양후중심부가조금융기된 5 mm 직경의거칠고건조하고황갈색을띄는집락을형성하였다 (Fig. 1A, B). 균은그람양성막대균으로 (Fig. 1C), 항산성염색 (Ziehl-Neelsen staining) 음성, 변법항산성염색에서는양성이었다 (Fig. 1D). 분리된균주는 API Coryne system와 50CH system (biomérieux Sa., Marcy-l Etoile, France) 을이용하여생화학적동정을시행하였다 (Table 1). 48시간후 API Coryne system에서 parazinamidase, alkaline phosphatase, α-glucosidase, catalase 양성이고, nitrate 환원, pyrolidonyl arylamidase, β-glucuronidase, β-galactosidase, N- acetyl-β-glucosaminidase 음성이고, esculin과 gelatin의가수분해음성 (profile , %ID = 85.0) 으로 Rhodococcus 속으로동정되었다. Christensen s urea agar에서는배양 96시간에양성반응을보였으며, esculin 가수분해는 7일까지음성이었다. 균종동정을위해 16S rrna 유전자염기서열분석을추가로실시하였다. 순수분리된집락에서 DNeasy Blood and Tissue Kit (QUIAGEN, Hilden, Germany) 로 DNA를추출하여 16S rrna의 번째염기를증폭하였다. 시발체는 54F (5 -AGA GTT TGA TCC TGG CTC AG-3 ), 1464R (5 -AAG GAG GTG ATC CAG CCG CA-3 ) 이었고, PCR 조건은 95 에서 30초간 DNA 변성 (denaturation), 58 에서 30초간복원 (annealing), 72 에서 2분간 DNA 연장 (extension) 과정을 35회반복하였다. 약 1,400 bp의 PCR 증폭산물을 QIAquick spin (QUIAGEN) 으로분리한후얻어진 16S Table 1. Comparison of the phenotypic characteristics of the clinical isolate with the characteristics of other clinically important Tsukamurella spp. Characteristics YMC11-5-B1957 T. inchonensis T. paurometabola T. pulmonis T. strandjordae T. tyrosinosolvens Growth at 35 C C + + Utilization of D-cellobiose + + NA D-maltose + + V + D-mannitol D-sorbitol NA + glycerol + V + V V V potassium 2 ketogluconate V V + xylitol + V V + V inositol α-methyl-d-glucoside + + V V D-melezitose + + V + D-arabinose + V V V D-ribose + V V arbutin V + V salicin + V + V L-fucose + V D-arabitol Hydrolysis of esculin NA + gelatin Presence of urease NA + catalase NA + nitrate reductase NA API-Coryne 48 hr days Abbreviations:, negative; +, positive; ND, not available; V, variable

4 rrna 염기서열을 EZtaxon server version 2.1에서 [15] 검색한결과, T. inchonensis (GenBank accession no. X ) 와가장높은상동성 (99.86%, 1402/1404 bp) 를보였고, 다음으로 T. paurometabola (GenBank accession no. Z ) 와 99.64% (1398/1403 bp) 의상동성을보였다. 높은상동성 ( 98%) 을보이는표준균주들의 16S rrna 염기서열과 MEGA 5.01 프로그램을이용하여 phylogentic tree를분석한결과 [16] T. inchonensis 으로동정할수있었다 (Fig. 2). Clinical Laboratory Standards Institute (CLSI) 의호기성방선균 (actinomycetes) 항균제감수성판독기준에따라 [17], E-test (AB BIODISK, Solna, Sweden) 와 VITEK2 P601 (biomerieux) 를이용하여항균제감수성을시험하였다. 항균제최소억제농도는 amikacin 2.0 μg/ml, cefepime 0.75 μg/ml, cefotaxime 1.0 μg/ml, trimethoprim-sulfamethoxazole 0.19 μg/ml, gentamicin 4 μg/ml, imipenem μg/ml, linezolid 8 μg/ml, clarithromycin 4.0 μg/ml, amoxicillin-clavulanic acid 32.0 μg/ml, ciprofloxacin 4.0 μg/ml, tobramycin 16.0 μg/ml로 amoxicillin-clavulanic acid, ciprofloxacin, tobramycin 에내성, clarithromycin 에중간, 그외모든시험항균제에감수성을보였다 (Table 2). 이후환아는두달간격으로두차례, 발열을주소로재입원하였고, 혈액배양에서지속적으로 T. inchonensis 가검출되었다. 환아는동일하게아미노글리코시드계, 세팔로스포린계, 메트로니다졸계의항균제를투여받고증상이호전되어각각입원 7일과 10일후에퇴원하였다. 첫입원에서넉달후의마지막입원에서중심정맥관을제거하였고, 일년후요로감염의심되어입원하여시행한혈액배양에서는더이상 T. inchonensis 가검출되지않았다. 고찰 Tsukamurella 속은 Nocardia, Gordonia, Streptomyces, Rhodococcus, Corynebacterium, Mycobacterium 속과함께 actinomycetes 에속하는편성호기성그람양성막대균으로토양이나절 지동물에서발견된다 [1, 8]. 완속발육균으로집락의모양이거친 점, 약한항산성염색상의특성등으로인해호기성방선균 (actinomycetes) 에속하는다른균속으로잘못동정될수있다. Shim 등은 Gram 염색, 항산성염색및생화학적성상을통해 T. pulmonis 를 Corynebacterium 속으로잘못보고한바있고 [8], Cho 등 Table 2. Antimicrobial susceptibility of a T. inchonensis isolate from a patient who was treated with antibiotics via an intravenous catheter Antimicrobials First drug Antimicrobial MIC (interpretation) of: YMC11-5-B1957 T. inchonensis (IMMIB D-771 T ) Amikacin 2 (S) 2 (S) Amoxicillin-clavulanic acid 32 (R) 128 (R) Ciprofloxacin 4 (R) 0.19 (S) Clarithromycin 4 (I) ND Imipenem (S) 0.38 (S) Linezolid 8 (S) ND Trimethoprim-sulfamethoxazole 0.19 (S) ND Tobramycin 16 (R) ND Second drug Cefepime 0.75 (S) ND Cefotaxime 1 (S) 1 (S) Gentamicin 4 (S) 6 (I) Abbreviations: ND, not determined; I, intermediate; R, resistant; S, susceptible; MIC, minimum inhibitory concentration. 97 Tsukamurella inchonensis IMMIB D-771T (X85955) 74 YMC11-5-B Tsukamurella paurometabola (AF283280) 70 Tsukamurella strandjordae ATCCBAA-173 (AF283283) Tsukamurella tyrosinosolvens DSM (AY238514) Tsukamurella carboxydivorans Y2 (EU521689) Tsukamurella spumae N1176 (AY238513) Tsukamurella pulmonis IMMIB D-1321T (X92981) Tsukamurella spongiae K362 (AY714239) Fig. 2. A dendrogram illustrating the phylogenetic relationships between our clinical isolate (YMC11-5-B1957) and T. inchonensis, T. paurometabola, T. tyrosinosolvens, T. strandjordae, T. pulmonis, T. carboxydivorans, T. pseudospumae, T. spumae, T. sunchonensis, and T. spongiae strains based on the 16S rrna gene sequencing. The scale indicates the similarity index

5 [12] 은 API Coryne system과 rpob 유전자의 PCR-RFLP 방법을이용하였을때 T. pulmonis 를비정형 Mycobacterium 으로잘못동정한예를보고한바있다. 이증례의경우상품화된동정 kit를사용하여 YMC11-5-B1957 를 Rhodococcus 속으로 1차동정하였으나, 추가로분자생물학적시험을시행하여 Tsukamurella 속으로재동정하였다. 환아는발열, 빌리루빈증가와간문-장문합술수술을받았던과거력을고려하여, 담낭염을의심하였고 cefpiramide, ami kacin, metronidazole 등으로경험적항균제투여후증상이소실되었다. 1차및 2차입원에서는중심정맥관 tip 배양을시행하지않았으나중심정맥관을유지한채로퇴원한이후두차례에걸쳐발열을주소로입원을반복하였다. 이때마다혈액배양에서지속적으로 T. inchonensis 가검출되었던점과중심정맥관을제거한이후는증상이소실된점, 그리고혈액배양에서균증식이검출되지않았던점을고려할때중심정맥관과관련된 T. inchonensis 패혈증으로판단하였다 [18]. Tsukamurella 속에의한감염은대부분정맥관이나인공삽입물과관련이있는것으로보고된다. 또한종양등의기저질환을가지고있거나투석을받는면역저하환자에서발생한다 [4, 5, 8-10]. 이증례의경우정맥관을유지하고있는점은기존보고들과유사하나 [10], 면역저하제를투여받지않은정상적인면역기능을갖는환아였다. 이분리주의생화학적성상을 T. inchonensis, T. paurometabola, T. pulmonis, T. strandjordae, T. tyrosinosolvens 와비교하였을때, 42 에서자란다는점에서 T. inchonensis 를제외한균종과차이가있었다 (Table 1). YMC11-5-B1957 의당이용능은 T. inchonensis 와가장유사한성상을보였으나, esculin 가수분해능음성으로새로운아종 (subspecies) 일가능성이있는것으로판단하였다. 16S rrna 유전자염기서열을분석한결과표준균주인 T. inchonensis IMMIB D7717 T (GenBank accession no. X ) 와 99.79%, T. paurometabola DSM (GenBank accession no. CP ) 와 99.57% 의일치도를보였다. CLSI MM-18[11] 의지침에따라판독하였을때 T. inchonensis 와 T. paurometabola 염기서열이 0.4% 이상의차이를보이지않아, T. paurometabola 의가능성을배제할수는없었으나, Genbank에등록된다른표준균주들과 phylogentic tree 분석을시행하였을때 [16], T. inchonensis 와동일한 branch를이룬것을확인하였다 (Fig. 2). 더군다나 YMC11-5-B1957 의생화학적반응양상이 T. paurometabola 와큰차이를보여서 (Table 1) T. inchonensis 로동정할수있었다. 호기성방선균은상품화된생화학적동정제품으로종동정을할수없기때문에이들균종에의함감염을진단하려면생화학적성상뿐아니라, 16S rrna 염기서열분석이필요한것으로판단하였다. Tsukamurella 속의항균제감수성검사는 CLSI의호기성방선 균 (actinomycetes) 기준에따른다. 이증례에서도기존의보고들과동일하게판독하였다 [8, 10]. Yassin 등 [19] 과 Chong 등 [20] 이보고한 T. inchonensis 와비교하였을때, amikacin, imipenem, cefotaxime에감수성, amoxicillin-clavulanic acid에내성인것은동일하였으나, 기존에감수성이던 ciprofloxacin 에내성이고중간이던 gentamicin에감수성을보이는차이점이있었다 (Table 2). Bouza 등 [10] 은 Tsukamurella 속의항균제감수성에결과가시험방법에따라큰차이를보일수있으며이균속에의한균혈증치료에있어항생제의유용성은아직불확실하다고하였고, 중심정맥관제거가 Tsukamurella 속혈류감염증의치료에효과적이라고보고하였다. 이증례도항균제치료에도불구하고, 두달후 Tsukamurella 속혈류감염증이다시검출되었고, 중심정맥관을제거한후에 T. inchonensis 균혈증이완전히소실되었다. 임상미생물검사실에서는중심정맥관을가진환자에서 coryneform 그람양성간균에의한균혈증이검출될때 Tsukamurella 균종을고려하여감별진단을수행하여야할것으로판단하였다. 요약 Tsukamurella 속은산소성으로약한항산성을띠며가지를치지않는그람양성막대균이다. Tsukamurella 균속이중심정맥관관련균혈증을일으킨예가보고되어있으며, Corynebacterium 이나비정형 Mycobacterium 로잘못동정되기도하므로이균종의감염은실제보다드물게진단될것으로추정한다. 따라서중심정맥관을가진환자에서혈액배양에서 coryneform 그람양성막대균이분리되어중심정맥관관련균혈증이의심될때 Tsukamurella 균종을감별진단에넣어야할것이다. 저자들은담도폐쇄증으로간문-장문합술수술을시행받은 4세환아에서중심정맥관과연관되어발생한 T. inchonensis 균혈증을보고하는바이다. 참고문헌 1. Conville PS and Witebsky FG. Nocardia, Rhodococcus, Gordonia, Actinomadura, Streptomyces, and Other Aerobic Actionmycetes. In: Murray PR, Baron EJ, et al., eds. Manual of clinical microbiology. 9th ed. Washington DC: ASM Press, 2007: Maalouf R, Mierau SB, Moore TA, Kaul A. First case report of community-acquired pneumonia due to Tsukamurella pulmonis. Ann Intern Med 2009;150: Alcaide ML, Espinoza L, Abbo L. Cavitary pneumonia secondary to Tsukamurella in an AIDS patient. First case and a review of the literature. J Infect 2004;49:

6 4. Larkin JA, Lit L, Sinnott J, Wills T, Szentivanyi A. Infection of a knee prosthesis with Tsukamurella species. South Med J 1999;92: Shaer AJ and Gadegbeku CA. Tsukamurella peritonitis associated with continuous ambulatory peritoneal dialysis. Clin Nephrol 2001;56: Woo PC, Ngan AH, Lau SK, Yuen KY. Tsukamurella conjunctivitis: a novel clinical syndrome. J Clin Microbiol 2003;41: Woo PC, Fong AH, Ngan AH, Tam DM, Teng JL, Lau SK, et al. First report of Tsukamurella keratitis: association between T. tyrosinosolvens and T. pulmonis and ophthalmologic infections. J Clin Microbiol 2009; 47: Shim HE, Sung H, Baek SM, Namgung S, Kim MN, Kim YG, et al. A case of catheter-related bacteremia of Tsukamurella pulmonis. Korean J Lab Med 2009;29: Elshibly S, Doherty J, Xu J, McClurg RB, Rooney PJ, Millar BC, et al. Central line-related bacteraemia due to Tsukamurella tyrosinosolvens in a haematology patient. Ulster Med J 2005;74: Bouza E, Pérez-Parra A, Rosal M, Martín-Rabadán P, Rodríguez-Créixems M, Marín M. Tsukamurella: a cause of catheter-related bloodstream infections. Eur J Clin Microbiol Infect Dis 2009;28: 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: Clinical and Laboratory Standards Institute, Cho AR, Kim HR, Lee MK, Choi SH, Yun SW. A case report of Tsukamurella pulmonis infection misidentified as atypical mycobacteria. Korean J Clin Microbiol 2010;13: Park JC, Hong J, Seo JG, Chung WK, Seo YH, Lee HH. A case of Tsukamurella tyrosinosolvens peritonitis associated with continuous ambulatory peritoneal dialysis. Korean J Med 2009;76: Lo SF. Reference intervals for laboratory tests and procedures. In: Kliegman R, Nelson WE, eds. Nelson textbook of pediatrics. 19th ed. Philadelphia, PA: Elsevier/Saunders, Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, et al. EzTaxon: a webbased tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol 2007;57: Saitou N and Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4: National Committee for Clinical Laboratory Standards. Susceptibility testing of Mycobacteria, Nocardiae, and other aerobic actinomycetes; approved standard. NCCLS document M24-A. Wayne, PA: National Committee for Clinical Laboratory Standards, Centers for Disease Control and Prevention: National Healthcare Safety Network. Guidelines and procedures for monitoring CLABSI (Central Line-Associated Bloodstream Infection). PDFs/pscManual/4PSC_CLABScurrent.pdf (Updated on Jun 2011). 19. Yassin AF, Rainey FA, Brzezinka H, Burghardt J, Lee HJ, Schaal KP. Tsukamurella inchonensis sp. nov. Int J Syst Bacteriol 1995;45: Chong Y, Lee K, Chon CY, Kim MJ, Kwon OH, Lee HJ. Tsukamurella inchonensis bacteremia in a patient who ingested Hydrochloric acid. Clin Infect Dis 1997;24:

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2186 대한진단검사의학회지제 29 권제 1 호 2009 Korean J Lab Med 2009;29:41-7 DOI 10.3343/kjlm.2009.29.1.41 Case Report Clinical Microbiology 중심정맥관관련 Tsukamurella pulmonis 균혈증 1 예 심효은 1 성흥섭 1 백승미 1 남궁승 1 김미나 1 김용균 2 이규형 2