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Korean J Clin Microbiol Vol. 12, No. 2, June, 2009 Septic Peripheral Embolism in Left Leg from Aggregatibacter aphrophilus Endocarditis Ja Young Lee 1, Si Hyun Kim 1, Haeng Soon Jeong 1, Seung Hwan Oh 1, Hye Ran Kim 1, Young Il Yang 2,4, Yang Haeng Lee 3, Jeong Nyeo Lee 1,4, Jeong Hwan Shin 1,4 Departments of 1 Laboratory Medicine, 2 Pathology, 3 Thoracic Surgery, 4 Paik Institute for Clinical Research, Inje University College of Medicine, Busan, Korea Aggregatibacter aphrophilus is a facultatively anaerobic gram-negative coccobacillus or bacillus that grows with no dependence on X factor and variable requirement for V factor. The organism is normal flora in the human oral cavity and upper respiratory tract and, rarely, causes invasive infections such as bacteremia, endocarditis, brain abscess, or osteomyelitis. We report a case of septic peripheral embolism in left leg from A. aphrophilus endocarditis. A 49-year-old man with known hypertension presented with acute muscle pain in the left leg. On physical examination, a regular heartbeat with a pansystolic murmur was heard. There were decreased pulses in the left popliteal and dorsalis pedis arteries and coldness of the left foot, although sensory and motor functions were intact. Angiography revealed an embolus in a branch of the left femoral artery. He underwent emergency embolectomy, and gram-negative bacilli grew in the embolus cultures. The same microorganism was isolated in two pairs of blood culturs and subsequently identified as A. aphrophilus. Transthoracic echocardiography revealed mitral regurgitation and multiple vegetations on the mitral valve. The patient was treated with a third-generation cephalosporin for 4 weeks and mitral valve replacement in view of the diagnosis of infective endocarditis and septic peripheral embolism. (Korean J Clin Microbiol 2009;12:82-86) Key Words: Embolism and thrombosis, Endocarditis, Aggregatibacter aphrophilus 서 Aggregatibacter는통성혐기성, 비운동성, 그람음성막대균혹은짧은막대균으로증식을위해 X인자 (hemin) 를요구하지않는반면 V인자 (nicotinamide adenine dinucleotide) 에대해다양한요구도를보이는균속으로 Aggregatibacter actinomycetemcomitans, Aggregatibacter aphrophilus, Aggregatibacter segnis가이에속한다 [1]. 정상인의구강과치태에서흔히분리되는구강점막정상균무리로주로치주염을유발하며드물게심내막염, 균혈증, 뇌농양, 골수염등을일으킨다 [2-4]. 감염성심내막염은판막손상, 전이성감염, 색전증등의합병증을동반할수있는데색전증은환자의예후에큰영향을미치기때문에빠른진단및치료가필요하다. 색전이주로침범하는부위는중추신경계로신경학적휴유증을남기며이외에 Received 15 December, 2008, Revised 7 April, 2009 Accepted 15 May, 2009 Correspondence: Jeong Hwan Shin, Department of Laboratory Medicine, Busan Paik Hospital, Inje University College of Medicine, 633-165, Gaegeum-dong, Busanjin-gu, Busan 614-735, Korea. (Tel) 82-51- 890-6475, (Fax) 82-51-893-1562, (E-mail) jhsmile@inje.ac.kr 론 도비장, 신장, 팔다리등을침범하는것으로알려져있다 [5,6]. Haemophilus spp. 는다른그람음성세균보다심초음파상증식물이커서색전증발생위험이높은데 [7], 가장흔한원인균인 Haemophilus parainfluenzae에의한심내막염에서색전증발생이주로보고되고있으며 A. aphrophilus 심내막염에서발생한말초동맥색전증은매우드물다 [5]. 이에저자들은하지동맥의색전제거술을시행받은환자의색전조직과 2쌍의혈액배양에서 A. aphrophilus를동정하고심초음파에서승모판막의다발성증식물을발견하여 A. aphrophilus 감염성심내막염에서발생한패혈성하지동맥색전증을진단한증례를보고하는바이다. 증례 49세남자환자가내원당일오전갑자기발생한왼쪽다리근육통증을주소로응급실을통해내원하였다. 환자는 6개월전에고혈압을진단받고약물치료를받아왔으며내원 2개월전부터발열, 기침및근육통이있었고 1개월전일차병원에서상기도감염을진단받았다. 환자는내원당시급성병색을보였으나의식은명료하였다. 내원시활력징후는혈압 130/80 82

Ja Young Lee, et al. : Peripheral Embolism by A. aphrophilus 83 mmhg, 심박수 110회 / 분, 호흡수 16회 / 분, 체온 37 C였다. 심음은규칙적이었으나범수축기잡음이청진되었고양측하지검사에서운동및감각은정상이었으나좌측슬와동맥과발등동맥의맥박이약하게촉지되었으며발에냉감이있었다. 내원당시일반혈액검사상백혈구 22,150 10 9 /L ( 호중구 92.2%) 로증가되었고, 혈색소 10.7 g/dl, 적혈구용적 27.5% 로감소되었으며혈소판수 248 10 9 /L로정상이었다. ESR 41 mm/hr, CRP 10.38 mg/dl 로증가되어있었으며혈액응고검사는정상이었다. 생화학검사상혈청총단백 8.8 g/dl, 알부민 2.8 g/dl으로 A/G 율이역전되어있었고, AST 101 U/L, ALT 113 U/L, 총빌리루빈 2.4 mg/dl, LDH 1,004 U/L로증가되어있었다. BUN 10 mg/dl로정상이었으나 creatinine 1.4 mg/dl 로약간증가되었고요검사에서 ph 5.5, 잠혈 250 이상, 요경검에서적혈구 51 100/μL가관찰되었다. 하지동맥의급성폐색증을의심하여실시한혈관조영술에서왼쪽온대퇴동맥과표재성대퇴동맥의분지부에서색전성폐색이관찰되어응급색전제거술을받았다 (Fig. 1). 적출된색전으로배양이의뢰되었고배양 1일만에혈액한천배지에서 1 mm 이하의작은이슬방울같은회백색집락이관찰되었다 (Fig. 2). 집락주위에용혈은관찰되지않았으며도말염색에서그람음성막대균이관찰되었다 (Fig. 3). Oxidase와 catalase 음성이었고 Vitek NHI card (biomeŕieux, Marcy l Etoile, France) 에서 95% Haemophilus aphrophilus/haemophilus paraphrophilus로동정되었다. 균동정을추가로확인하기위해 27F (5 -AGA GTT TGA TYM TGG CTC AG-3 ) 와 1492R (5 -GGY TAC CTT GTT ACG ACT T-3 ) 시발체를이용하여중합효소연쇄반응을실시하였으며증폭된약 1,500 bp 크기의산물에 대하여 27F (5 -AGA GTT TGA TYM TGG CTC AG-3 ), 515R (5 -TTA CCG CGG CKG CTG GCA C-3 ), 533F (5 -GTG CCA GCM GCC GCG GTA A-3 ), 926F (5 -AAA CTY AAAKGA ATT GAC GG-3 ), 1050R (5 -CAC GAG CTG ACG ACA-3 ), 1492R(5 -GGY TAC CTT GTT ACG ACT T-3 ) 을이용하여 16S rdna 염기순서분석을시행하였다. 16S rdna 염기순서분석결과 A. aphrophilus (GenBank accession no. M75041) 와 99.5% 일치도를보였고차상위로 Aggregatibacter segnis (M75043) 와 97.4% 일치도를보였다. X, V인자요구도검사를위해 35 C에서 24시간배양한결과분리균주는 X, V인자디스크에상관없이광범위한세균증식을보였으며, catalase 음성, lactose, sucrose에서산생성양성등의 Fig. 2. Gray-whitish colored pinpoint colonies with no hemolysis growing on sheep blood agar plate after 24-hour incubation at 35 C in 5% CO 2. Fig. 1. Angiogram shows embolic occlusion of left femoral artery in bifurcation region between common femoral and superficial femoral arteries. Fig. 3. Microscopy of embolus cultures shows gram-negative bacilli (Gram stain; 1,000).

84 Korean J Clin Microbiol 2009;12(2):82-86 생화학반응과 16S rdna 염기서열분석결과 A. aphrophilus 로최종동정하였다. 내원당시응급실에서실시한 2쌍의혈액배양에서도그람음성막대균이증식하였고색전배양에서분리된균주와같은생화학성상을보여상기방법과동일한방법으로동정한결과 A. aphrophilus로확인되었다. 패혈성말초동맥색전증원인을찾기위해실시한경흉부심초음파에서승모판전, 후엽의다발성증식물 (0.46 0.23 cm, 1.12 0.79 cm, 0.79 0.27 cm) 및 grade III 승모판역류가관찰되어 A. aphrophilus에의한감염성심내막염을진단하였다. 색전의기타장기침범여부를알아보기위해실시한뇌자기공명영상검사에서색전에의한다발성뇌경색소견이관찰되었다. Cefolatam으로치료를시작한후일주일뒤에시행한혈액배양에서균은더이상자라지않았고, 25병일에승모판막치환술을받았다. 술후 2일째실시한심초음파에서인공판막기능은양호하였으며이후퇴원하여 warfarin 복용하면서경과관찰중이다. 고찰 Haemophilus 중에서 H. aphrophilus와 H. paraphrophilus는주요생화학반응이동일하여상품화된미생물동정기기에서같은동정결과를보이며 16S rdna 염기순서분석을이용한분자학적검사에서도 DNA 유사성이매우높아정확한감별이어렵다 [8-10]. 이두균주의주요감별점인 X, V인자요구도검사를통한감별이필요한데 H. paraphrophilus는성장에 V인자를필요로하며 H. aphrophilus는 X, V인자를요구하지않는특징을가지고있다. Nørskov-Lauritsen 과 Kilian[1] 은 H. aphrophilus의 V인자독립성이 nadv (nicotinamide phosphoribosyltransferase) 유전자와연관되며 H. paraphrophilus는 H. aphrophilus의 DNA가 NAD 독립적인표현형으로전환되어 V인자요구성을가지는것으로보았다. 따라서 H. paraphrophilus를 H. aphrophilus의 heterotypic synonym으로간주하고두균주를합쳐 A. aphrophilus로명명하였다. 본증례의분리균주는생화학적성상및 16S rdna 염기순서분석을통해 A. aphrophilus로동정되었으며 X, V인자요구도검사결과 X, V인자에상관없이광범위한증식을보였다. A. aphrophilus에의한심내막염은주로발치와같은치과적처치, 구강외상, 치과질환및상기도감염등의선행력을가진환자에서발생한다 [11-13]. 본증례환자의경우내원전치과치료를받은병력은없었으나수술전검사에서만성치주염을진단받았고내원 1개월전부터상기도감염치료를받은과거력을가지고있어심내막염과연관성을추정할수있었다. 색전증은감염성심내막염에서심부전에이어두번째로치명적인합병증으로주로중추신경계를침범하여신경학적후 유증을남기거나비장, 신장등주요장기에농양을형성하며팔다리동맥을침범하는경우는빈도가낮다 [14]. 색전증은감염성심내막염진단전, 진단후항균제치료중혹은치료가끝난후에도발생할수있으나대부분감염성심내막염진단후항균제치료첫 2주에발생하며 [15] 본증례처럼감염성심내막염을진단받기전첫증상으로말초동맥색전증이발생한보고는매우드물다. 급성팔다리허혈증상이발생하는경우대부분동맥경화에의한혈전증으로진단하기쉬우나본증례의환자처럼상기도감염선행력이있거나치주질환이있는경우감염성심내막염에의한패혈성색전증가능성을고려하여야한다. 또한이경우색전증의재발위험이높기때문에적절한항균제투여및수술을통해원인이되는판막의증식물을반드시제거하여야한다 [15]. Vilacosta 등 [15] 은증식물이승모판에있으면서크기가 1 cm 이상일경우색전증발생빈도가현저히증가한다고보고하였으며본증례의환자도심초음파상증식물이승모판전, 후엽에다발성으로존재하였고가장큰증식물의크기가 1.12 0.79 cm로색전증발생의고위험군임을확인할수있었다. 감염균에따른색전증발생빈도를살펴보면감염성심내막염의주요원인균주인 Staphylococcus spp. 에서색전증발생률이가장높게보고되었으며 [16], 그람음성세균에서는 Haemophilus spp. 가다른그람음성세균보다심초음파상판막의증식물이크고색전증발생도더흔한것으로알려져있다 [7]. 국내에서는현재까지보고되어있는 A. aphrophilus 심내막염중 2 예에서뇌자기공명영상검사상뇌농양및색전이관찰되었고 ampicillin 및 3세대 cephalosporin 투여를통해완치되었다 [13,17]. A. aphrophilus에의한심내막염치료시과거에는 penicillin 유도체와 aminoglycoside 병합요법이일반적이었으나 β-lactamase 를생성하는균종의출현으로현재는 3세대 cephalosporin을 4 6주간투여하는것이표준으로되어있다 [18]. 본증례의균주는 β-lactamase 음성이었고환자는균동정후 4주간 3세대 cephalosporin을투여받았으며승모판막치환술을받았다. 항균제치료일주일후시행한혈액배양은음성이었으며적출된승모판막에서는미세혈전과함께유리질변성이관찰되었다. 본증례는하지동맥의색전제거술을시행받은환자의색전조직과 2쌍의혈액배양에서 A. aphrophilus를동정하고심초음파에서승모판막의다발성증식물을발견하여 A. aphrophilus 감염성심내막염에서발생한패혈성하지동맥색전증을진단하였던증례이다.

Ja Young Lee, et al. : Peripheral Embolism by A. aphrophilus 85 참고문헌 1. Nørskov-Lauritsen N and Kilian M. Reclassification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov., and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V factor-independent isolates. Int J Syst Evol Microbiol 2006;56:2135-46. 2. Huang ST, Lee HC, Lee NY, Liu KH, Ko WC. Clinical characteristics of invasive Haemophilus aphrophilus infections. J Microbiol Immunol Infect 2005;38:271-6. 3. Colson P, La Scola B, Champsaur P. Vertebral infections caused by Haemophilus aphrophilus: case report and review. Clin Microbiol Infect 2001;7:107-13. 4. Kao PT, Tseng HK, Su SC, Lee CM. Haemophilus aphrophilus brain abscess: a case report. J Microbiol Immunol Infect 2002;35: 184-6. 5. Darras-Joly C, Lortholary O, Mainardi JL, Etienne J, Guillevin L, Acar J. Haemophilus endocarditis: report of 42 cases in adults and review. Haemophilus Endocarditis Study Group. Clin Infect Dis 1997;24:1087-94. 6. Groeneveld JH and Puylaert JB. Images in clinical medicine. Septic peripheral embolization from Haemophilus aphrophilus endocarditis. N Engl J Med 2002;347:816. 7. Parker SW, Apicella MA, Fuller CM. Hemophilus endocarditis. Two patients with complications. Arch Intern Med 1983;143:48-51. 8. Dogǎn B, Asikainen S, Jousimies-Somer H. Evaluation of two commercial kits and arbitrarily primed PCR for identification and differentiation of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus. J Clin Microbiol 1999;37:742-7. 9. Dix K, Watanabe SM, McArdle S, Lee DI, Randolph C, Moncla B, et al. Species-specific oligodeoxynucleotide probes for the identification of periodontal bacteria. J Clin Microbiol 1990;28:319-23. 10. Tønjum T, Bukholm G, Bøvre K. Identification of Haemophilus aphrophilus and Actinobacillus actinomycetemcomitans by DNA- DNA hybridization and genetic transformation. J Clin Microbiol 1990;28:1994-8. 11. Akhondi H and Rahimi AR. Haemophilus aphrophilus endocarditis after tongue piercing. Emerg Infect Dis 2002;8:850-1. 12. Brouqui P and Raoult D. Endocarditis due to rare and fastidious bacteria. Clin Microbiol Rev 2001;14:177-207. 13. Kim CK, Cho I, Park YH, Roh KH, Yong D, Lee K, et al. Isolation of Haemophilus aphrophilus and coagulase-negative staphylococi from the blood of a patient with prosthetic valve endocarditis. Korean J Clin Microbiol 2006;9:71-5. 14. Bayer AS, Bolger AF, Taubert KA, Wilson W, Steckelberg J, Karchmer AW, et al. Diagnosis and management of infective endocarditis and its complications. Circulation 1998;98:2936-48. 15. Vilacosta I, Graupner C, San Romań JA, Sarria C, Ronderos R, Fernańdez C, et al. Risk of embolization after institution of antibiotic therapy for infective endocarditis. J Am Coll Cardiol 2002;39:1489-95. 16. Mu gge A, Daniel WG, Frank G, Lichtlen PR. Echocardiography in infective endocarditis: reassessment of prognostic implications of vegetation size determined by the transthoracic and the transesophageal approach. J Am Coll Cardiol 1989;14:631-8. 17. Cha CH, Shin HB, Jang S, Kim MK, Kim YS, Song JK, et al. A case of Haemophilus aphrophilus endocarditis. Korean J Clin Microbiol 2003;6:172-6. 18. Almeda FQ, Tenorio AR, Barkatullah S, Parrillo JE, Simon DM. Infective endocarditis due to Haemophilus aphrophilus treated with levofloxacin. Am J Med 2002;113:702-4.

86 Korean J Clin Microbiol 2009;12(2):82-86 = 국문초록 = Aggregatibacter aphrophilus 심내막염에서발생한패혈성하지동맥색전증 1 예 인제대학교의과대학 1 진단검사의학교실, 2 병리학교실, 3 흉부외과학교실, 4 백인제기념임상의학연구소이자영 1, 김시현 1, 정행순 1, 오승환 1, 김혜란 1, 양영일 2,4, 이양행 3, 이정녀 1,4, 신정환 1,4 Aggregatibacter aphrophilus는통성혐기성그람음성짧은막대균혹은막대균으로증식시 X인자를요구하지않고 V인자에다양한요구도를갖는특징을가지고있다. 구강및상기도정상균무리로드물게균혈증, 심내막염, 뇌농양, 골수염등의감염을일으키는것으로알려져있다. 저자들은 A. aphrophilus 심내막염에서발생한패혈성하지동맥색전증 1예를보고하는바이다. 6개월전고혈압을진단받은 49세남자환자가내원당일갑자기발생한왼쪽다리통증을주소로내원하였다. 이학적검사상심음은규칙적이었으나범수축기잡음이청진되었으며양측하지검사에서운동및감각은정상이었으나좌측슬와동맥과발등동맥의맥박이약하게촉지되었으며발에냉감이있었다. 혈관조영술상왼쪽대퇴동맥의분지에색전성폐색이관찰되어응급색전제거술을시행받았으며적출된색전배양에서그람음성막대균이증식하였다. 같은날실시한 2쌍의혈액배양에서도동일균이증식하였고이균은 A. aphrophilus로최종동정되었다. 말초동맥색전증원인을찾기위해실시한경흉부심초음파상승모판에다발성증식물및승모판역류가관찰되어감염성심내막염및패혈성하지동맥색전증진단하에 4주간 3세대 cephalosporin을투여하였고승모판막치환술후퇴원하였다. [ 대한임상미생물학회지 2009;12:82-86] 교신저자 : 신정환, 613-735, 부산시부산진구개금동 633-165 인제대학교의과대학부산백병원진단검사의학교실 Tel: 051-890-6475, Fax: 051-893-1562 E-mail: jhsmile@inje.ac.kr