증례보고 Lab Med Online Vol. 8, No. 1: 34-38, January 2018 임상미생물학 Dysgonomonas capnocytophagoides 에의한담도성패혈증 1 예 A Case of Biliary Sepsis by Dysgonomonas capnocytophagoides 민성희 1 이혜영 1 장정현 1 성흥섭 1 김미나 1 * 배미현 2 김명환 3 Sunghee Min, M.D. 1, Hye-Young Lee, M.D. 1, Jeong-Hyun Chang, M.D. 1, Heungsup Sung, M.D. 1, Mi-Na Kim, M.D. 1 *, Mi-Hyun Bae, M.D. 2, Myung-Hwan Kim, M.D. 3 울산의대서울아산병원진단검사의학과 1, 한양대학교구리병원진단검사의학과 2, 울산의대서울아산병원소화기내과 3 Department of Laboratory Medicine 1, University of Ulsan College of Medicine and Asan Medical Center, Seoul; Department of Laboratory Medicine 2, Hanyang University Guri Hospital, Hanyang University College of Medicine, Guri; Division of Gastroenterology, Department of Internal Medicine 3, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea Dysgonomonas capnocytophagoides is a gram-negative, facultatively anaerobic coccobacillus that was formerly designated CDC group dysgonic fermenter (DF)-3, occurring as a normal flora in human gut and rarely causing human infections such as bacteremia, abscess, diarrhea, and cholecystitis. In this study, we report a case of biliary sepsis caused by D. capnocytophagoides in a patient with biliary obstruction. A seventy fouryear-old man, admitted to the hospital due to common bile-duct stone, also had cholangitis caused by D. capnocytophagoides and Enterococcus avium, which were isolated from his blood cultures. D. capnocytophagoides was initially identified as D. gadei by MALDI-TOF mass spectrometry, but later confirmed as D. capnocytophagoides by 16S rrna gene sequencing. To the best of our knowledge, this is the first report of human infection by D. capnocytophagoides in Korea. Key Words: Dysgonomonas capnocytophagoides, Sepsis, Cholelithiasis 서론 Dysgonomonas capnocytophagoides 는조건무산소성 (facultative anaerobe) 그람음성알막대균이다. Dysgonomonas spp. 는 2000 년에 Hofstad 등에의해처음명명되었으며 [1], 그전까지는 CDC group DF-3 로분류되어있었다 [2]. 최근까지 D. gadei, D. capnocytophagoides, D. mossii, D. hofstadii, D. oryzarvi, D. macrotermitis, D. termitidis, D. alginatilytica 등 8 종이보고되었으며 [3], Dysgonomonas 속에의한인체감염은매우드물게보고되었 Corresponding author: Mi-Na Kim Department of Laboratory Medicine, Asan Medical Center, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea Tel: +82-2-3010-4511, Fax: +82-2-478-0884, E-mail: mhkim@amc.seoul.kr Received: May 31, 2017 Revision received: August 26, 2017 Accepted: September 5, 2017 This article is available from http://www.labmedonline.org 2018, Laboratory Medicine Online This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 다. D. mossii와 D. gadei가각각담낭염환자의담낭에서발견된증례가보고되었으며 [1, 4], D. capnocytophagoides가면역억제환자에서설사를일으킨증례와급성골수성백혈병환자에서치료중균혈증을일으킨증례가보고되었다 [4, 5]. 국내에서 Dysgonomonas 속에의한인체감염이보고된적은없다. 저자들은담석에의한담도폐쇄환자에서 Dysgonomonas capnocytophagoides 에의한패혈증증례를경험하여문헌고찰과함께보고하고자한다. 증례 고혈압외특이병력이없는 74세남자환자가 2일간지속된열과우상복부통증으로응급실로내원하였다. 응급실에서시행한 CT 에서여러개의총담관결석이발견되었으며, 간내담도와간외담도가모두확장되어있는소견을보여총담관결석에의한담도폐쇄와그로인한간담도염으로진단받았다. 입원당시환자의활력징후는혈압 108/63 mmhg, 맥박 107회 / 분, 호흡 23회 / 분, 체온 37.2 C 이었으며, 혈액검사결과는백혈구 20.6 10 3 /μl ( 호중구 92.9%, 림프구 4.7%, 단핵구 2.0%, 호산구 0.4%), 총빌리루빈 4.2 mg/dl, C- 반응단백 13.07 mg/dl였다. 입원당일시행한담즙그람염색에서 34 www.labmedonline.org eissn 2093-6338
백혈구가 10-25/LPF 로관찰되었으며, 그람양성알균이관찰되었다. 환자는입원당일피부간경유쓸개관배액관 (percutaneous transbiliary drainage, PTBD) 과내시경역행쓸개이자조영술 (ERCP) 을통해내시경코쓸개배액관 (ENBD) 을설치하였다. 패혈성쇼크를의심하여내과계중환자실로입원하였다. 입원당일 meropenem을투여하였고, 내원 2일째부터 6일까지 imipenem과 teicoplanin으로치료하였으나, 백혈구 14.8 10 3 /μl, 총빌리루빈 2.1 mg/dl, C-반응단백 8.31 mg/dl로패혈증소견과담도폐쇄증상이지속되었다. 이후내원 7일째에다시 ERCP를시행하여일부담석을제거하였으며, 항생제를 imipenem과 metronidazole로변경하였다. 내원 10 일째추적 ERCP를시행하여남은담석을모두제거하였고, 내원 14일째 PTBD를제거한후백혈구 4.1 10 3 /μl, 총빌리루빈 0.7 mg/dl, C-반응단백 0.1 mg/dl로증상호전되어퇴원하였다. 환자는입원당일담즙과혈액에서배양을진행하였으며, 이후추가적인추적배양은진행하지않았다. 담즙배양에서 Enterococcus avium, Escherichia coli, Citrobacter amalonaticus가배양되었다. 입원당일말초정맥에서채취한 3쌍의혈액배양은각각 Bactec Plus Aerobic/F 병과 Lytic/10, Anaerobic/F (Becton Dickinson, Sparks, MD, USA) 병에접종하여 Bactec FX 시스템 (Becton Dickinson) 에서배양하였다. 말초정맥에서채취한 3쌍의혈액배양중 2 쌍의혈액배양에서는무산소병에서각각 30.1시간과 76.1시간후에그람음성알막대균이검출되었고 (Fig. 1A), 산소병에서는균이검출되지않았다. 나머지 1쌍은산소병에서그람음성알막대균이 49.9시간후에검출되었으며, 무산소성병에서그람양성알균이 32.3 시간후에검출되었다. 그람양성알균은 Enterococcus avium으로동정되었다. 혈액에서분리된 E. avium과담즙에서분리된 E. avium 은동일한항균제감수성을보여같은균으로판단되었다. 분리된그람음성알막대균은혈액한천배지에서는자랐으나, MacConkey 배지에서는자라지않았다 (Fig. 1B). 그람음성알막대균에대해 MALDI-TOF mass spectrometry (Bruker Daltonics Inc., Billerica, MA, USA) 를이용하여동정을시행하였고, 1.815점으로 Dysgonomonas gadei라는결과를주었으며, 다음순위는 1.275점으로 Neisseria gonorrhoeae이었다. MALDI-TOF 점수가 2.0 이하였고, 검사실에서처음분리된드문균종이었기때문에, 정확한균종확인을위해 16S rrna 유전자염기서열분석을실시하였다. 순수분리된집락에서 GenElute Bacterial Genomic DNA 키트 (Sigma, St. Louis, MO, USA) 로 DNA를추출한후 16S rrna의 8-806번째염기와 515-1,390번째염기부위를각각증폭하였다. 시발체의염기서열은 8FPL 5 -AGTTTGATCCTGGCTCAG- 3, 806R 5 -GGACTAC- CAGGGTATCTAAT- 3, 515FPL 5 -TGCCAGCAGCCGCGGTAA-3, 13B5 -AGGCCCGGGAACGTATTCAC-3 이었으며, PCR 조건은기존문헌을따랐다 [6]. 증폭산물은 Power Gel Extraction kit (Ta- KaRa Bio Inc., Shiga, Japan) 로정제한후마크로젠 (Seoul, Korea) 에염기서열분석을의뢰하였다. 16S rrna 염기서열 BLAST database (http://www.ncbi.nlm.nih.gov/blast/) 에서검색한결과 D. capnocytophagoides JCM 16697 (AB548674) 와 99.70% (1,336/1,340), D. alginatilytica HUA-2 (LC021528) 와 95.09% (1,277/1,343) 의일치도를보여, D. capnocytophagoides로판정할수있었다 [7]. D. capnocytophagoides는 oxidase 검사와 catalase 검사는음성, Triple Sugar Iron agar test에서사면과바닥층이산성이고, gas 생성과황화수소생성은없었다. Bile esculin 가수분해검사에서양성으로 bile 내성이있었다. 분리된 D. capnocytophagoides 의 A B Fig. 1. Microscopic findings and colony morphology of Dysgonomonas capnocytophagoides. (A) Gram-negative coccobacilli visualized under the microscope after 30.1 hr in the anaerobic culture vial (Gram stain, 1,000 ). (B) Grey-white, smooth, and non-hemolytic colonies grew on blood agar, but none on MacConkey agar. www.labmedonline.org 35
생화학적특성을보기위하여 API 20E (biomérieux, Durham, NC, USA) 와 API 20NE (biomérieux) 로추가적인생화학검사를진행하였고, acetoin production, gelatin hydrolysis, glucose fermentation, sucrose, melibiose, amygdalin, arabinose, esculine ferric citrate, β-galactosidase에양성, mannitol, inositol, sorbitol, rhamnose, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urea hydrolysis, tryptophan deamination, indole production 에서음성이었다. 5% 면양적혈구를첨가한 Mueller Hinton 한천배지에서 E-test strip (Biodisk, Solna, Sweden) 을사용하여항균제감수성검사를시행하였다. 최소억제농도는 meropenem >32 μg/ml, gentamicin >32 μg/ml, benzylpenicillin >32 μg/ml, metronidazole 0.5 μg/ ml, ertapenem >32 μg/ml, clarithromycin 12 μg/ml, tetracycline 0.19 μg/ml, colistin 0.016 μg/ml, ceftriaxone >32 μg/ml, tigecycline 0.023 μg/ml, imipenem >32 μg/ml이었다. 디스크확산법감수성검사에서 ciprofloxacin, gentamycin, imipenem, cefepime, penicillin, cefazolin, tobramycin, cefoxitin 에서는억제대가관찰되지않았으며, tetracycline은 32 mm, trimethoprim-sulfamethoxazole 은 30 mm의억제대가관찰되었다. 고찰 이증례는국내에서첫 Dysgonomonas spp. 감염보고이다. Dysgonomonas spp. 는 CDC group DF-3에속하는균으로 2000년에 Dysgonomonas로새로이명명되었으며, 인체감염을일으키는것으로알려졌다 [1, 4, 8]. 이균종들은담관및담낭관련감염과면역억제환자에서의균혈증, 면역억제환자에서의설사를일으킨증례가보고되었다 [1, 4, 8-10]. D. capnocytophagoides로인한균혈증은 Hansen 등 [4] 과 Hironaga 등 [11] 이각각호중구감소성발열환자와췌장암환자에서보고하였으며, CDC group DF-3와다른 Dysgonomonas spp. 도주로면역저하환자에서설사, 균혈증, 농, 담낭염등을일으키는것으로보고되었다 [1, 5, 9, 10, 12]. 하지만이증례는평소고혈압약을복용하는것이외에는건강하여정상적인면역상태였다. 이는 Dysgonomonas spp. 가면역저하환자에서뿐만아니라비면역저하환자에서도심각한감염을일으킬수있음을시사한다. 이증례는입원당시응급실에서시행한말초혈액배양검사에서각각무산소성병에서는 30.1, 76.1시간후에, 산소성병에서는 49.9 시간후에검출되어 3쌍의혈액에서모두 D. capnocytophagoides 가자랐으며, 이중 1쌍에서 E. avium이함께자랐으므로 D. capnocytophagoides와 E. avium에의한패혈증으로진단할수있었다. 말초혈액배양에서분리된것과별개로같은날 PTBD를통해 얻은담즙배양에서는 D. capnocytophagoides 가분리되지않고, E. avium, E. coli, C. amalonaticus 등 3 종의장내세균총이자랐 으며, 이중 E. avium 은혈액배양과담즙배양둘다에서분리되었 다. 이는 Hironaga 등 [11] 이담도염환자의혈액배양에서무산소병 에서 D. capnocytophagoides 가자랐다고보고하는등 Dysgonomonas spp. 는무산소성환경을선호하는것으로판단된다. 따라 서이증례에서담즙배양은산소성환경에서만시행하였기때문 에 D. capnocytophagoides 를분리하지못했을가능성이있다. Dysgonomonas spp. 는담즙에내성이있어서담도계에서집락을 형성할수있다는증거들이있다 [1, 12, 13]. D. gadei 가처음분리 된곳이사람의담낭이었고 [1], D. mossii 도담도배액에서분리된 바있다 [12]. 더욱이 D. capnocytophagoides 는담도염이발생한췌 장암환자에서균혈증을일으킨보고가있어서담도폐쇄가 Dysgonomonas spp. 균혈증의위험요인일것으로추정할수있다 [11]. 이증례또한담도폐쇄가있는환자로담즙배양에서분리되지는 않았지만, D. capnocytophagoides 가 E. avium 과함께담도성패 혈증을일으킨것으로판단하였다. 증례에서분리된 D. capnocytophagoides 는생화학검사에서 Table 1. Biochemical test responses of the current isolate, compared to those of D. capnocytophagoides and Serratia plymuthica Response of Biochemical test D. capnocytophagoidesthica Serratia plymu- This case Oxidase Negative Negative Negative β-galactosidase Positive Positive Positive Arginine dihydrolase Negative Negative Negative Lysine decarboxylase Negative Negative Negative Ornithine decarboxylase Negative Negative Negative H2S production Negative Negative Negative Urea hydrolysis Negative Negative Negative Tryptophan deamination Negative Negative Negative Indole production Negative Negative Negative Acetoin production Positive Negative Positive Gelatin hydrolysis Positive Negative Positive Carbohydrate fermentation Glucose Positive Positive Positive Mannitol Negative Negative Positive Inositol Negative Negative Negative Sorbitol Negative Negative Negative Rhamnose Negative Negative Negative Sucrose Positive Positive Positive Melibiose Positive Positive Positive Amygdalin Positive Positive Positive Arabinose Positive Positive Positive *CCUG 17996 T (LMG 11519T) [1]; API 20E biomerieux database (http://210.242.211.31/ jsp/ident/index.jsp API 20E V5.0); Require differential diagnosis among API 20 E database. 36 www.labmedonline.org
bile esculin 가수분해검사에서양성으로기존보고처럼 bile에내성이있었다. Acetoin production, gelatin hydrolysis 에서기존보고와달리양성이었으나나머지생화학적반응은일치하였다 [1, 4, 14]. 이러한생화학적반응양상은 apiweb 데이터베이스 (http://210. 242.211.31/jsp/ident/index.jsp, API 20E V5.0) 에서는 mannitol fermentation을제외하면 Serratia plymuthica 와가장일치도가높았고, 50.6% 로 S. plymuthica로동정하였다 (Table 1). 하지만 D. capnocytophagoides는 apiweb 데이터베이스에없기때문에 API 만으로정확한균종동정을하기힘들다. Dysgonomonas spp. 의생화학적특성이기존의보고와일부에서다른결과를보이는등생화학적특성이균주에따라변화가있을수있고, 상품화된제품을사용시 database에 Dysgonomonas spp. 가포함되지않기때문에다른균종으로동정될수있다는것을보여준다. 따라서 Dysgonomonas spp. 의동정에는생화학적검사를이용한동정보다는분자생물학적검사가필요하다고판단된다. MALDI-TOF를이용한동정에서는 D. gadei로동정되었며, 1.815 log 스코어값을보였다. 이는 MALDI-TOF의데이터베이스에 Dysgonomonas spp. 로는 D. gadei만있고, D. capnocytophagoides 는없기때문에 D. gadei가비교적높은점수를보인것으로판단된다. 이러한점들때문에 Dysgonomonas spp. 처럼드물고 MALDI-TOF 점수가 2.0 미만이면서 MALDI-TOF의 database가해당속에속하는균종을충분히포함하지못하는경우에는균종에대한분자생물학적확진검사가필요할것으로생각된다. Dysgonomonas spp. 의항균제감수성에대한자료는부족한편이다. 본증례에서분리된 D. capnocytophagoides는항균제감수성검사에서 metronidazole, tetracycline, colistin, tigecycline, trimethoprim-sulfamethoxazole에감수성이있었으며, imipenem, meropenem, ciprofloxacin과 cephalosporin 계열의항균제에대하여내성을가지고있었다. 증례의환자는 imipenem 과 teicoplanin 으로 3일간경험적항균제치료후, 배양결과에따라항균제를 imipenem과 metronidazole로변경하였으며, 이후증상이호전되었다. Dysgonomonas spp. 는 trimethoprim-sulfamethoxazole, clindamycin, tetracycline, metronidazole 등에공통적으로감수성을보이며, ampicillin, meropenem, imipenem, aminoglycosides에는예측할수없는감수성을보이고, cephalosporin 계열과 fluoroquinolone 계열의항균제에대해서는내성을보인다 [1, 4, 12]. 이증례에서분리된 D. capnocytophagoides는이런감수성양상과잘일치하였다. Dysgonomonas spp. 에대한표준적인항균제치료지침이없고, β-lactams 계열의항균제감수성은예측할수없기때문에심각한감염의원인이라면명확한균종동정과함께추가적인항균제감수성검사가필요할것으로생각된다. 이증례는 D. capnocytophagoides에의한패혈증의국내첫보 고이다. 담석으로인한담도폐쇄후발생한패혈증으로발생하였으며, ERCP와 imipenem, metronidazole 등항균제로치료하였다. 담도폐쇄는 Dysgonomonas spp. 에의한패혈증의위험요인으로추정되며, 균종동정을위해서는생화학적검사보다분자생물학적인검사가유용할것이다. Carbapenem 계열의항균제에내성을가질수있어서패혈증등심각한감염시에는항균제감수성검사를실시하는것이권장된다. REFERENCES 1. Hofstad T, Olsen I, Eribe ER, Falsen E, Collins MD, Lawson PA. Dysgonomonas gen. nov. to accommodate Dysgonomonas gadei sp. nov., an organism isolated from a human gall bladder, and Dysgonomonas captocytophagoides (formerly CDC group DF-3). Int J Syst Evol Microbiol 2000;50:2189-95. 2. Wallace PL, Hollis DG, Weaver RE, Moss CW. Characterization of CDC group DF-3 by cellular fatty acid analysis. J Clin Microbiol 1989;27:735-7. 3. Kita A, Miura T, Okamura Y, Aki T, Matsumura Y, Tajima T, et al. Dysgonomonas alginatilytica sp. nov., an alginate-degrading bacterium isolated from a microbial consortium. Int J Syst Evol Microbiol 2015;65: 3570-5. 4. Hansen PS, Jensen TG, Gahrn-Hansen B. Dysgonomonas capnocytophagoides bacteraemia in a neutropenic patient treated for acute myeloid leukaemia. APMIS 2005;113:229-31. 5. Wagner DK, Wright JJ, Ansher AF, Gill VJ. Dysgonic fermenter 3-associated gastrointestinal disease in a patient with common variable hypogammaglobulinemia. Am J Med 1988;84:315-8. 6. Relman DA. Universal bacterial 16S rrna amplification and sequencing. In: Persing DH, Smith TF, et al. eds. Diagnostic molecular microbiology principles and applications. 1st ed. Washington DC: ASM Press, 1993:489-95. 7. 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, 2008. 8. Gill VJ, Travis LB, Williams DY. Clinical and microbiological observations on CDC group DF-3, a gram negative coccobacillus. J Clin Microbiol 1991;29:1589-92. 9. Blum RN, Berry CD, Phillips MG, Hamilos DL, Koneman EW. Clinical illnesses associated with isolation of dysgonic fermenter 3 from stool samples. J Clin Microbiol 1992;30:396-400. 10. Heiner AM, DiSario JA, Carroll K, Cohen S, Evans TG, Shigeoka AO. www.labmedonline.org 37
Dysgonic fermenter-3: a bacterium associated with diarrhea in immunocompromised hosts. Am J Gastroenterol 1992;87:1629-30. 11. Hironaga M, Yamane K, Inaba M, Haga Y, Arakawa Y. Characterization and antimicrobial susceptibility of Dysgonomonas capnocytophagoides isolated from human blood sample. Jpn J Infect Dis 2008;61:212-3. 12. Matsumoto T, Kawakami Y, Oana K, Honda T, Yamauchi K, Okimura Y, et al. First isolation of Dysgonomonas mossii from intestinal juice of a patient with pancreatic cancer. Arch Med Res 2006;37:914-6. 13. Kodama Y, Shimoyama T, Watanabe K. Dysgonomonas oryzarvi sp. nov., isolated from a microbial fuel cell. Int J Syst Evol Microbiol 2012; 62:3055-9. 14. Zbinden R. Aggregatibacter, Capnocytophaga, Eikenella, Kingella, Pasteurella, and other fastidious or rarely encountered gram-negative rods. In: Jorgensen JH, Pfaller MA, Carroll KC, Landry ML, Guido Funke, Richter SS, et al. ed. Manual of Clinical Mcirobiology, 11th ed. Washington, DC. Amercian Society for Microbiology 2015. 652-66. 38 www.labmedonline.org