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1 Biomedical Science Letters 2019, 25(3): 275~281 eissn : Original Article Genetic Diversity of Metallo-β-lactamase Genes of Chryseobacterium indologenes Isolates from Korea Jong Hwa Yum,* Department of Clinical Laboratory Science, Dongeui University, Busan 47340, Korea This study was performed to characterize the chromosomal metallo-β-lactamases (MBLs) of Chryseobacterium indologenes isolated from Korea and to propose a clustering method of IND MBLs based on their amino acid similarities. Chromosomal MBL genes were amplified by PCR from 31 clinical isolates of E. indologenes. Nucleotide sequencing was performed by the dideoxy chain termination method using these PCR products. Antimicrobial susceptibilities were determined by the agar dilution method. PCR experiments showed that all 31 E. indologenes isolates contained all bla IND genes. DNA sequence analysis revealed that E. indologenes isolates possessed ten types of bla IND gene, including seven novel variants (bla IND-8 to bla IND-14 ). The most common combination of MBL was IND-2 (n = 18). Minimum inhibitory concentrations of imipenem and meropenem for the isolates harboring novel IND MBLs were 16 μg/ml. IND MBLs were grouped in three clusters, based on amino acid similarities. Key Words: Chryseobacterium indologenes, Metallo-β-lactamase, IND, bla IND, Cluster 서론 Chryseobacterium indologenes 는물이나토양과같은자연에흔히분포하고있지만, C. indologenes 는원내감염이나기회감염을통해다양한임상증상을나타내는것으로알려져있다 (Hsuh et al., 1997; Kirby et al., 2004; Atıcı et al., 2016; Rabenandrasana et al., 2019). C. indologenes 는 imipenem 과 meropenem 과같은 carbapenem에대해내성을보일수있는 metallo-β-lactamase (MBL) 을가지고있어대부분의 β-lactams 제제에대해내성을보인다 (Bellais et al., 2000; Yu et al., 2015). 또한, C. indologenes 는흔히다약제내성을보여치료시약제선택에어려움이있고, 치사율도 14% 에달한다 (Hsueh et al., 1997; Douvoyiannis et al., 2010). C. indologenes 의 ambler class B MBL에속하는 indologe- nease (IND) 를최초로프랑스의 Bellais 등이보고하였고 (Bellais et al., 1999), 이후, 2000 년에 Bellais 등이임상에서분리한 C. indologenes 에서 bla IND-1~4 를검출하여보고하였다 (Bellais et al., 2000). 2008년에는중국에서 Lin 등이 1,500 병상대학병원에서분리균주에서검출한 bla IND-1~3 을보고한바있고, 대부분이 imipenem 과 meropenem 에높은항균제최소억제농도 (Minimal inhibitory concentration; MIC) 를보였다 (Lin et al., 2008). 이태리의 Perilli 등은 2007년 pi. 8.8의 IND-5 를보고하였다 (Perilli et al., 2007). 2009년이태리의또다른병원에서검출된 IND-6를 Zeba 등이검출하였는데, ceftazidime 과 cefepime 을잘분해하지못하는 IND-2와 IND-5와는달리다양한 β-lactamase 제제를비교적효율적으로분해하였다 (Zeba et al., 2009) 년일본에서 Yamaguchi 등은 IND-7 을검출하였고, 이효소의 X-ray 에의한분자구조를밝혔다 (Yamaguchi et al., 2010). 임상에서분리되는 C. indologenes 의염색체성 bla IND 는시기와지 Received: August 15, 2019 / Revised: September 2, 2019 / Accepted: September 9, 2019 * Professor. Corresponding author: Jong Hwa Yum. Department of Clinical Laboratory Science, Dongeui University, Busan 47340, Korea. Tel: , Fax: , auxotype@deu.ac.kr C The Korean Society for Biomedical Laboratory Sciences. All rights reserved. CC 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

2 역에따라다양한특성을보이며검출되는것을알수있다. 따라서본연구에서는국내대학병원환자로부터분리된 C. indologenes 의 MBL의종류와유전자특성을규명하고자하였다. 재료및방법시험균주신촌세브란스병원과개금백병원에서 1994년부터 2009년까지분리된 C. indologenes 31주를대상으로하였으며, 균종동정은전통적방법과 16S rdna의염기서열분석을이용하였다 (Loffler et al., 2000). 본연구에서사용한임상분리균주는 20% skim milk 에부유하여 -70 에보관하여시험하였다. Escherichia coli ATCC 와 Pseudomonas aeruginosa ATCC 27853을항균제최소억제농도시험에참조균주로사용하였다. 항균제감수성시험항균제감수성시험은 CLSI (Clinical and Laboratory Standards Institute, 2017) 의권고에따라 10 4 colony forming units의접종액을 Mueller-Hinton agar (Becton Dickinson, Sparks, MD., USA) 에접종하여고체한천희석법으로결정하였다. 시험에사용한항균제는 amoxicillin (Kun Wha Pharmaceuticals, Seoul, Korea), piperacillin (Wyeth, Pearl River, NY, USA), cephalothin (Sigma Chemical Co., St. Louis, USA), ceftazidime (GlaxoSmithKline, Greenford, UK), cefotaxime (Handok, Seoul, Korea), cefoxitin 과 imipenem (Merck Sharp & Dohme, Rahway, NJ., USA), aztreonam 과 cefepime (Bristol- Myers Squibb, Plainsboro, NJ., USA), moxalactam (Eli Lilly & Co., Indianapolis, IN., USA), 그리고 meropenem (Sumitomo, Tokyo, Japan) 이었다. MBL 표현형검출 MBL 생성균주와 MBL 비생성균주를구별하기위해 Lee 등이보고한방법으로 Hodge 변법과 imipenem-edta 변법 + sodium mercaptopropionic acid (SMA) double disc synergy (DDS) 시험을시행하였다 (Lee et al., 2004). 분자생물학적분석염색체유래 IND MBL을암호화하는유전자검출은 Table 1에기술한시발체를이용하여 PCR 증폭을시행하였다. PCR은 premix taq polymerase (Bioneer, Cheongwon, Korea), 각각의시발체는 20 pmol, 열에의해추출된주형 DNA 1 μl를첨가하고, 최종양을 100 μl로조정하여시행하였다. PCR 반응조건은 94 5분간주형 DNA를 denature 시키고, 이후 94 30초, 50 30초, 72 60초로 35회반응하고, 마지막 72 7분간 extension 반응하였다. PCR 생성물은염기서열분석하였다. 모든 PCR 생성물양가닥은 automatic sequencer (ABI3700: Perkin-Elmer, Foster City, CA., USA) 로 2회염기서열분석하였다. bla IND 유전자의 open reading frame (ORF) 의 5' 와 3' 말단염기서열은 Sørensen 등이제시한방법과같이 simple two-step PCR로 Table 1. The primers used for detection or sequencing Primer 5'-to-3'sequence Product size (bp) Reference INDD-F INDD-R INDS-F INDS-R N1INDS-F N1INDS-R NN2INDS-F NN2INDS-R N2IND-seq-R N3INDS-F N3INDS-R 16SRNA-F 16SRNA-R 5'-GCC CAG GTT AAA GAT TTT GTA AT-3' 5'-CAT GGC CAC CGC CTT TCC ATT C-3' 5'-GGT TTG CAT ATC TAT CTG CC-3' 5'-ATC CAA AGA GAG GCT GGA GT-3' 5'-CTT TAA TTA CTA CCT TAA AAA TT-3' 5'-AAT ACA AAT CCC GGA TGA C-3' 5'-TTT AAT TAT TAC CCT AAA AAT T-3' 5'-AAA TCC CGA ATT TTC ATT CG-3' 5'-CCC AGG GAA CAT CAA ACA G-3' 5'-AAC TTT AAT TAC TGC TTT AAA TT-3' 5'-TTT CAA TAC TGC GGA AGA G-3' 5'-AGA GTT TGA TCC TGG CTC AG-3' 5'-AAG GAG GTG ATC CAG CCG CA-3' Steinberg et al., This study 802 This study 845 Used for sequencing This study 850 This study 1,544 Loffler et al., 2000

3 시행하였다 (Sørensen et al., 1993). 이들 MBL의아미노산서열은프로그램 CLUSTAL X version 2.0 (ftp://ftp-igbmc.- ustrasbg.fr/pub/clustalx) 로분자생물학적유연관계를분석하였다. Nucleotide sequence accession numbers 본연구에서밝힌새로운 bla IND 염기서열은 GenBank nucleotide database 에다음의 accession number로등록하였다. GU (bla IND-2c ), GU (bla IND-7 ), GU (bla IND-8 ), GU (bla IND-9 ), GU (bla IND-10 ), HM (bla IND-11 ), HM (bla IND-12 ), HM (bla IND-13 ), HM (bla IND-14 ). 결과임상분리주특성임상에서분리된 imipenem 내성 C. indologenes 31주모두는 Hodge 변법과 imipenem-edta+sma DDS 시험양 Table 2. Clinical characteristics of specimens with isolates of C. indologenes in Korean hospitals Strains No. Specimen bla IND Underlying disease YMC94/01/3047 Sputum bla IND-7 Cervical cord tumor YMC94/04/3376 Sputum bla IND-8 Cerebral infarction YMC94/12/3839 Sputum bla IND-7 Aspiration pneumonia YMC96/09/3073 Sputum bla IND-9 Subdural hematoma YMC96/09/3300 Sputum bla IND-9 Oropharyngeal abscess YMC03/04/833 Urine bla IND-2b Tuberculous spondylitis YMC03/04/558 Sputum bla IND-2 Venococclusive disease DEUIJM Sputum bla IND-2c Pulmonary tuberculosis preterm infants DEUIJM Blood bla IND-10 Aplastic anemia DEUIJM Sputum bla IND-2a Pulmonary embolism DEUIJM Sputum bla IND-2 Atherosclerosis DEUIJM Blood bla IND-2c Lung cancer DEUIJM Sputum bla IND-2a Pulmonary tuberculosis preterm infants DEUIJM Blood bla IND-2c Viral encephalitis DEUIJM Cerebrospinal fluid bla IND-2 Intracerebral hemorrhage DEUIJM Blood bla IND-11 Parkinson's disease DEUIJM Cerebrospinal fluid bla IND-13 Encephalomyelitis DEUIJM Sputum bla IND-2c Liver cirrhosis DEUIJM Sputum bla IND-2c Cerebral infarction DEUIJM Sputum bla IND-13 Pulmonary tuberculosis preterm infants DEUIJM Sputum bla IND-2c Intracerebral hemorrhage DEUIJM Bronchial washing bla IND-2c Bacterial pneumonia DEUIJM Sputum bla IND-2c Cerebral infarction DEUIJM Sputum bla IND-2c Lung cancer DEUIJM Sputum bla IND-2c Subarachnoid hemorrhage DEUIJM Sputum bla IND-14 Mitral stenosis DEUIJM Sputum bla IND-2b Klatskin tumor DEUIJM Sputum bla IND-2b Preterm infants DEUIJM Catheter tip bla IND-2b Preterm infants DEUIJM Sputum bla IND-2b Preterm infants DEUIJM Sputum bla IND-12 Extreme immaturity

4 성으로 MBL 생성주이었다. 이들 MBL 생성 C. indologenes 있어 IND-2와 함께 다른 cluster로 분류되었다(Fig. 1). C. 31주 중 22주가 객담에서 분리되어 가장 많았고, 이들 환 indologenes 9주의 IND 아미노산 서열의 alignment를 시행 자는 주로 면역저하 환자이었으며, 31명 중 7명이 사망 한 결과 73~100%의 identity를 보였다. 하였다(Table 2). 다음으로 MBL 생성 C. indologenes가 많 IND-1 cluster에는 IND-3, IND-5, IND-6, IND-7, IND-8, 이 분리된 검체는 혈액으로 4주가 분리되었으며, 그 외 검체에서는 5주가 분리되었다. Metallo-β-lactamase의 유전형 임상에서 분리된 31주 C. indologenes 모두는 MBL을 생 성하였다. 모든 C. indologenes 분리주는 PCR에 의해 IND 를 암호화하는 유전자가 검출되었다. C. indologenes 분리 주는 DNA 염기서열 분석에서 새로 밝혀진 7종의 새로운 변이종(blaIND-8에서 blaind-14까지)을 포함하여 8종의 IND 유전형을 나타냈다(Table 2). C. indologenes 31주 중 IND-2 를 생성하는 균주는 18주로 가장 흔하였고, 9주에서 새로 운 blaind alleles가 검출되었다. Metallo-β-lactamase의 유전적 유연관계 Phylogenic 연구에서 13종의 IND 효소는 아미노산 서 열에 따른 유사도를 근거로 3가지 cluster로 그룹지어지 는 것으로 나타났다(Fig. 1). Bellais 등(Bellais et al., 2000)이 프랑스에서 처음 보고한 IND-4를 제외한 IND-1 type과 IND-2 type의 cluster는 각각 유전적으로 가까운 것으로 보 인다. 한편, 본 연구에서 시험한 균주의 IND 10종의 유전 형은 2가지의 cluster에 속하였다. 이들 중 IND-11~13은 기존에 보고된 다른 IND와 아미노산 서열이 다소 차이가 Fig. 1. Dendrogram obtained for 14 representative INDs calculated with CLUSTALX version 2.0. The red circle ( ) indicates novel IND enzymes. Fig. 2. Comparison of amino acid sequence of the IND β-lactamase from C. indologenes isolates. The hyphen (-) indicates identical amino acid sequence. The asterisk (*) and cross (+) indicate the residues known to be involved either directly or indirectly with Zn1 and Zn2 cofactors, respectively

5 MBL types (No. of isolates) Table 3. MICs of β-lactams for IND type β-lactamase-producing clinical isolates of C. indologenes in Korean hospitals MIC range (µg/ml) a IPM MEM AZT CEP CTX CAZ FEP MOX FOX PIP IND-2 (18) 32~>128 16~>128 32~>128 >128 32~>128 4~64 4~128 32~128 8~128 4~>256 IND-7 (2) 16~32 32~64 32~64 > ~64 64~128 64~ ~32 IND-8 (1) > > IND-9 (2) 16~128 16~ >128 32~>128 4~ ~32 4~64 IND-10 (1) > IND-11 (1) > IND-12 (1) > IND-13 (4) 32~64 16~32 64~>128 >128 32~64 4~32 16~>128 64~ ~16 IND-14 (1) > Total (31) 16~>128 16~>128 16~>128 >128 16~>128 2~64 0.5~>128 32~128 8~128 4~>256 a IMP, imipenem; MEM, meropenem; AZT, aztreonam; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; CEP, cephalothin; MOX, moxalactam; FOX, cefoxitin; AMX, amoxicillin; PIP, piperacillin IND-9, IND-10, 그리고 IND-14 효소가포함된다 (Fig. 1). IND-2 cluster 에는 IND-11, IND-12, 그리고 IND-13 효소가포함된다. IND-4 cluster는 IND-1 cluster와 IND-2 cluster 와는진화적으로유연관계가먼것으로나타났고, 새로운 IND로밝혀진 IND-11, IND-12, 그리고 IND-13 간에는유연관계가가까운것으로나타났다. Class B β-lactamases 에대한 standard numbering scheme 에따른, Zn1의 ligand로예상되는 His 116, His 118, 그리고 His 196 의아미노산잔기와 Zn2의 ligand 로예상되는 Asp 120, Cys 221, 그리고 His 263 의아미노산잔기는 7종의새로운 IND 효소에서도잘보존되어있었다 (Fig. 2). 항균제감수성시험 β-lactam 제의 MIC는 piperacillin, ceftazidime 및 cefepime 을제외하고는대부분비교적높았는데, 이는 Zeba 등의 (Zeba et al., 2009) 보고와비슷하였다 (Table 3). Imipenem 과 meropenem MICs 는 C. indologenes 에대해서모두 16 μg /ml이었고, IND-9~12 생성균주는 ceftazidime 과 cefepime 의 MICs는각각 2 μg/ml 과 0.5 μg/ml 으로비교적낮았다. 고찰본연구결과국내에서분리된 C. indologenes 는모두 bla IND 를갖고있었다. 프랑스의 Bellais 등이 1999년처음보고한 C. indologenes 의 IND-1 이후로 7종의 IND 효소가 보고되었다 (Bellais et al., 1999; Bellais et al., 2000; Perilli et al., 2007; Lin et al., 2008; Zeba et al., 2009; Yamaguchi et al., 2010). 본연구에서는국내 3차대학병원에서분리한 C. indologenes 에서 9종의 IND를검출했고, 이들균종에서 carbapenem 내성에관여할것으로예상되는새로운 7종의 IND 효소를갖는균종에대해 imipenem 과 meropenem 의 MIC 값이 16 μg/ml 이상으로나타나이들균주모두내성이었다. C. indologenes 은기저질환이있는면역저하환자에서치명적인감염을일으킬수있는것으로알려져있다 (Nulens et al., 2001; Lin et al., 2003; Bayraktar et al., 2007; Hossam et al., 2007; Douvoyiannis et al., 2010). 본연구에서도높은치사율을보였다 (23%, n = 7). C. indologenes 감염시치료방법은다양하게알려져있으나, imipenem 이나 meropenem 과같은 carbapenem 뿐아니라다양한항균제에대한내성을보이는이들감염치료에필요한지침확립이필요해보인다. 결론적으로, 국내대학병원에서분리된 C. indologenes 는모두 bla IND 를갖고있었고, 이들중 9주는 7가지의새로운 bla IND 를갖고있었다. IND는아미노산서열의유사도에근거해볼때, 7종의새로운변이효소를포함해서 3개의 cluster 로나뉘며, 이들유전자를가진세균은 carbapenem 사용의증가로원내에서점차증가할것으로판단된다

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7 Yamaguchi Y, Takashio N, Wachino J-I, Yamagata Y, Arakawa Y, Matsuda K, Kurosaki H. Structure of metallo-β-lactamase IND-7 from a Chryseobacterium indologenes clinical isolate at 1.65-Å resolution. J Biochem : Yu CY, Ang GY, Cheng HJ, Cheong YM, Yin WF, Chan KG. Draft genome sequence of a multidrug-resistant Chryseobacterium indologenes isolate from Malaysia : Zeba B, Luca FD, Dubus A, Delmarcelle M, Simpore, Nacoulma OG, Rossolini GM, Frere J-M, Docquier J-D. IND-6, a highly divergent IND-type metallo-β-lactamase from Chryseobac- terium indologenes strain 597 isolated in Burkina Faso. Anti- microb Agents Chemother : Cite this article as: Yum JH. Genetic Diversity of Metalloβ-lactamase Genes of Chryseobacterium indologenes Isolates from Korea. Biomedical Science Letters :

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