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Availability of MADLDI-TOF MS for Identification of Gram Positive Bacilli Isolated from Blood Culture
Biomed Sci Letters 2018;24:108-115
Published online June 30, 2018;  https://doi.org/10.15616/BSL.2018.24.2.108
© 2018 The Korean Society For Biomedical Laboratory Sciences.

Jin-Un Choi1,§, Sang-Ha Kim2,§, Su-Jeong Hwang3, Young-Bin Yu5, Sunghyun Kim4,†, and Young-Kwon Kim5,†

1Department of Laboratory Medicine, Chonnam National University Hospital, Gwangju 61469, Korea,
2Department of Laboratory Medicine, Konyang University Hospital, Daejeon 35365, Korea,
3Department of Dental Hygiene, College of Medical Sciences, Konyang University, Daejeon 35365, Korea,
4Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 46252, Korea,
5Department of Biomedical Laboratory Science, College of Medical Sciences, Konyang University, Daejeon 35365, Korea
Correspondence to: Sunghyun Kim. Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 46252, Korea. Tel: +82-51-510-0560, Fax: +82-51-510-0568, e-mail: shkim0423@cup.ac.kr
Young-Kwon Kim. Department of Biomedical Laboratory Science, College of Medical Sciences, Konyang University, Daejeon 35365, Korea. Tel: +82-42-660-6371, Fax: +82-42-543-6370, e-mail: ykkim3245@konyang.ac.kr
Received March 5, 2018; Revised April 25, 2018; Accepted May 17, 2018.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract

In the present study, results of the identification of Gram-positive bacilli (GPB) were analyzed by using the MALDITOF MS technique to score each 2-year blood culture at a university hospital. In addition, 16S rRNA sequence analyses and MALDI-TOF MS results are compared to targeting strains that had been isolated two or more times within the same patient, to evaluate the usefulness of MALDI-TOF MS in GPB identification. According to the cut-off (≥ 1.7) criteria, there were 410 (57.5%) reliable strains and 303 (42.5%) non-identified strains among the GPB identification results of 713 strains, using a microflex MALDI Biotyper (Bruker Daltonik GmbH, Bremen, Germany). The isolation appeared most often in the following order: Corynebacterium striatum, Bacillus cereus, Bacillus subtilis, Paenibacillus urinalis, and Listeria monocytogenes. Nearly three-fourths, 66 out of 89 (74.2%) of the strains for Corynebacterium striatum; 44 out of 60 (73.3%) strains for Bacillus cereus; and all (25 out of 25, 100%) Listeria monocytogenes strains were identified by their high scores of 2.0 or higher. Most (293 strains out of 303) non-identified strains were strains isolated only once and not significant as infectious bacilli. A total of 43 out of 50 (86.0%) strains matched and were able to be identified based on the 16 rRNA sequencing comparison results of strains that were isolated twice or more within the same patient and significant as infection bacilli. Non-matching among 5 out of 7 strains was not identified, even with MALDI-TOF MS. In conclusion, GPB can be identified in blood cultures using MALDI-TOF MS. This can be done accurately with ease, rapidly, and at a low cost. It is also thought to be helpful in GPB diagnosis and treatment.

Keywords : Blood culture, Gram positive bacilli, MADLDI-TOF MS, 16S rRNA sequencing
꽌濡

理쒓렐 쓽猷뚭린愿쓽 엫긽誘몄깮臾쇨궗떎뿉꽌뒗 Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)瑜 湲곕컲쑝濡 븳 썝씤洹 룞젙 떆뒪뀥쓽 룄엯씠 利앷븯怨 엳뒗 긽솴씠떎. 씠 寃궗踰뺤 엫긽寃泥대줈遺꽣 遺꾨━ 諛곗뼇맂 떒씪 吏묐씫쓣 씠삩솕븯뿬 吏꾧났愿뿉꽌 寃異쒓린뿉 룄떖븯뒗 떆媛꾩쓣 洹쇨굅濡 援ъ꽦 臾쇱쭏쓽 吏덈웾쓣 痢≪젙븯뒗 諛⑸쾿쑝濡 룞젙 떆媛꾩 洹좎< 떦 룊洹 빟 6遺 젙룄 냼슂맂떎(Holland et al., 1996). 룞젙쓣 쐞븳 鍮꾩슜 긽뭹솕맂 吏꾨떒 궎듃瑜 룷븿빐 쟾넻쟻 룞젙 諛⑸쾿(Seng et al., 2009)쓽 빟 20~30% 젙룄濡 誘몄깮臾 寃궗떎뿉꽌 씠슜븯湲곗뿉 媛꾪렪븯怨, 룞젙뿉 냼슂릺뒗 떆媛 삉븳 留ㅼ슦 吏㏃븘 留ㅼ슦 寃쎌젣쟻씤 諛⑸쾿쑝濡 븣젮졇 엳떎(Bizzini et al., 2010; Stevenson et al., 2010).

洹몃엺 뼇꽦 留됰洹(Gram positive bacilli, GPB)쓽 遺遺꾩 二쇰줈 솚寃쎌뿉꽌 議댁옱븯硫 鍮꾨퀝썝꽦쑝濡 븣젮졇 엳뿀湲 븣臾몄뿉, 洹 룞븞 엫긽誘몄깮臾쇨궗떎뿉꽌뒗 씠瑜 삤뿼洹좎쑝濡 媛꾩<븯뿬 젙솗븳 룞젙쓣 떆뻾븯吏 븡뒗 寃껋씠 씪諛섏쟻씠뿀떎(Funke et al., 1997). 븯吏留, GPB 以 紐뉖챺 蹂묒썝꽦 꽭洹좎쥌 룞젙쓣 븯뒗 寃껋 엫긽쟻쑝濡 以묒슂븯떎. 듅엳, 理쒓렐 硫댁뿭 븯옄굹 泥대궡 궫엯湲곌뎄瑜 泥섏튂븯뒗 솚옄媛 利앷븯怨 엳쑝硫, 빆깮젣 궗슜 삉븳 利앷븿뿉 뵲씪 GPB뿉 쓽븳 湲고쉶媛먯뿼씠 利앷븯怨 엳뒗 異붿꽭씠硫, GPB쓽 젙솗븳 룞젙 븘슂꽦씠 몢릺怨 엳쑝硫, 씠瑜 넻빐 媛먯뿼쓽 젙솗븳 썝씤 遺꾩꽍씠 媛뒫븯怨, 쟻젅븳 移섎즺瑜 쐞빐 겙 룄쓣 以 닔 엳쓣 寃껋쑝濡 뿬寃⑥쭊떎(Adderso et al., 2008).

씪諛섏쟻쑝濡 삁븸 諛곗뼇(Blood culture)쑝濡쒕꽣 遺꾨━릺뒗 GPB뒗 鍮꾩슜쓽 슚슜쟻씤 痢〓㈃뿉꽌 룞씪 솚옄뿉꽌 2쉶 씠긽 遺꾨━맆 寃쎌슦뿉留 젣븳쟻쑝濡 썝씤洹 룞젙쓣 떆뻾븯吏留, 湲곗〈뿉 엫긽誘몄깮臾쇨궗떎뿉꽌 떆뻾븯怨 엳뒗 썝씤洹 룞젙 諛⑸쾿 留롮 떆媛꾩씠 냼슂릺怨, 빆긽 룞씪븳 寃곌낵瑜 굹궡吏 븡뒗떎(Barberi et al., 2004). 뵲씪꽌, 16S rRNA 뿼湲곗꽌뿴 遺꾩꽍쓣 씠슜븳 遺꾩옄쑀쟾븰쟻 寃궗 諛⑸쾿쓣 씠슜븯뿬 솗씤 룞젙쓣 떆뻾븯怨, 씠瑜 몴以踰(gold standard method)쑝濡 룄엯븯怨 엳吏留, 씠 寃궗踰뺤 鍮꾩슜怨 떆媛꾩씠 留롮씠 냼슂맂떎뒗 젣븳젏씠 엳떎.

MALDI-TOF MS 湲곕컲 썝씤洹 룞젙 떆뒪뀥쓣 씠슜븳 湲곗〈쓽 留롮 뿰援щ뱾 遺遺 엫긽誘몄깮臾쇨궗떎뿉꽌 二쇰줈 遺꾨━릺뒗 洹몃엺 뼇꽦 븣洹(Gram positive cocci, GPC), 洹몃엺 쓬꽦 留됰洹(Gram negative bacilli, GNB)쓣 몴쟻쑝濡 븯怨 엳쑝硫, 씪遺뒗 GPB瑜 긽쑝濡 吏꾪뻾릺뿀떎(Carbonnelle et al., 2012; Dubois et al., 2012). 뵲씪꽌, 蹂 뿰援ъ뿉꽌뒗 援궡쓽 븳 븰蹂묒썝쓽 삁븸 諛곗뼇쑝濡쒕꽣 遺꾨━ 諛곗뼇맂 GPB瑜 긽쑝濡 MALDI-TOF MS瑜 떎떆븯뿬 삁븸 諛곗뼇뿉꽌 遺꾨━맂 GPB쓽 룞젙 洹좎< 닔 誘몃룞젙 洹좎< 닔瑜 鍮꾧탳빐 蹂댁븯怨, 룞씪 솚옄뿉꽌 2쉶 씠긽 遺꾨━맂 洹좎<瑜 긽쑝濡 16S rRNA 뿼湲곗꽌뿴 遺꾩꽍쓣 븿猿 떎떆븯뿬 洹 寃곌낵瑜 鍮꾧탳븿쑝濡쒖뜥, GPB瑜 룞젙븿뿉 엳뼱꽌 MALDI-TOF MS瑜 湲곕컲쑝濡 븳 썝씤洹 룞젙 떆뒪뀥쓽 쑀슜꽦쓣 룊媛빐蹂닿퀬옄 븯떎.

옱猷 諛 諛⑸쾿

엫긽寃泥

2014뀈 10썡遺꽣 2016뀈 9썡源뚯 援궡쓽 븳 븰蹂묒썝뿉꽌 삁븸 諛곗뼇씠 쓽猶곕맂 엫긽寃泥대줈遺꽣 遺꾨━ 諛곗뼇맂 GPB 珥 713二쇰 긽쑝濡 뿰援щ 떎떆븯떎.

삁븸 諛곗뼇

삁븸 諛곗뼇 궛냼꽦 諛곗(BacT/ALERT FA Plus, FA BACTEC TM Plus Aerobic/F), 臾댁궛냼꽦 諛곗(BacT/ALERT Standard Anaerobic, SN BACTEC TM Plus Anerobic/F)뿉 젒醫낇븯怨 媛곴컖 BacT/ALERT 3D Blood Culture System (bioMérieuxInc., Durham, NC, USA)怨 BD BACTEC™ FX blood culture system (BD, Spark, MD)뿉 5씪媛 諛곗뼇븯떎. 뼇꽦씠 굹삩 삁븸 諛곗뼇 蹂묒쓣 5% Sheep Blood Agar Plate (HANIL KOMED)뿉 젒醫낇븯뿬 5% CO2, 35°C 諛곗뼇湲곗뿉꽌 24떆媛 삉뒗 48떆媛 諛곗뼇븯떎.

MALDI-TOF MS System쓣 씠슜븳 洹몃엺 뼇꽦 留됰洹 (GPB)쓽 룞젙

GPB濡 솗씤맂 洹좎<瑜 긽쑝濡 Microflex MALDI Biotyper (Bruker Daltonik GmbH, Bremen, Germany) 옣鍮꾨줈 寃궗瑜 닔뻾븳 썑, MALDI Biotyper RTC software (V. 3.1)瑜 씠슜빐 寃곌낵瑜 遺꾩꽍븯뿬 꽭洹 醫낆쓣 룞젙븯떎. 吏곸젒 룄留먮쾿 븯猷삳갇 諛곗뼇븳 떊꽑븳 吏묐씫쓣 硫멸퇏 Wooden applicator瑜 씠슜빐 MSP 96 target polished steel BC microscout Target plate (Bruker Daltonics)뿉 룄留먰븯怨, 꽭洹좎씠 留덈Ⅸ 썑 留ㅽ듃由뒪 슜븸(50% acetonitrile, 2.5% trifluoroacetic acid)뿉 룷솕맂(α-cyano-4-hydroxycinnamic acid)쓣 뜑븳 떆빟 1 μL瑜 媛븯怨, 떎삩뿉꽌 셿쟾엳 嫄댁“떆궓 썑 microflex MALDI Biotyper 옣鍮꾩뿉 옣李⑺븯떎. Bacterial test standard (BTS)瑜 궗슜븯뿬 옣鍮 Calibration쓣 떎떆븯怨, m/z 2,000~20,000 踰붿쐞뿉꽌 痢≪젙맂 Mass spectra 룞젙 寃곌낵뒗 MALDI Biotyper RTC software (V. 3.1)瑜 씠슜빐 遺꾩꽍븯떎. 옣鍮 쉶궗뿉꽌 젣떆븯뒗 몴以 湲곗쓣 湲곕컲쑝濡 븯뿬 Cut-off score媛 2.0 씠긽씠硫 洹 醫 룞젙, 1.7 씠긽씠硫댁꽌 2.0 誘몃쭔씤 寃쎌슦 洹 냽 룞젙씠 媛뒫븳 寃껋쑝濡 뙋떒븯怨, 1.7 誘몃쭔씤 寃쎌슦 떊猶곗꽦씠 뾾뒗 寃껋쑝濡 媛꾩<븯떎.

16S rDNA 뿼湲곗꽌뿴 遺꾩꽍

Genomic DNA 異붿텧: 삁븸 諛곗뼇쑝濡쒕꽣 遺꾨━ 諛곗뼇맂 GPB쓽 gDNA 異붿텧 Phosphate buffered saline (PBS) 1 mL뿉 吏묐씫 3~4媛쒕 샎긽떆궓 썑 8,000 x G뿉꽌 5遺꾧컙 썝떖遺꾨━ 븯떎. 移⑥궗痢듭쓣 젣쇅븳 긽痢듭븸쓣 젣嫄고븳 썑 Lysis solution (DW 1 L, Tris base 1.212 g, EDTA 0.372 g, Triton 1 mL/1 L) 200 μL瑜 泥④븯怨 洹좎쓣 샎긽븳 썑 8,000 x G뿉꽌 5遺꾧컙 썝떖遺꾨━ 븯떎. Lysis 怨쇱젙쓣 3쉶 諛섎났 썑, 硫멸퇏 DW 200 μL뿉 移⑥궗痢듭쓣 샎긽떆궓 썑 100°C뿉꽌 30遺 룞븞 媛뿴븯뿬 gDNA媛 쑀由щ릺룄濡 븯떎. 씠썑 13,000 x G, 4°C뿉꽌 10遺꾧컙 썝떖遺꾨━ 븯뿬 긽痢듭븸쓣 痍⑦븳 썑 궗슜 쟾源뚯 -20°C뿉꽌 깋룞蹂닿 븯怨, 뿼湲곗꽌뿴 遺꾩꽍쓣 쐞븳 PCR 닔뻾 떆 gDNA쓽 냽룄瑜 100 ng뿉 留욎텛뼱 궗슜븯떎.

16S rDNA 뿼湲곗꽌뿴 遺꾩꽍쓣 쐞븳 PCR 諛 뿼湲곗꽌뿴 遺꾩꽍: 16S rDNA 뿼湲곗꽌뿴 遺꾩꽍쓣 쐞븳 PCR쓣 닔뻾븯湲 쐞빐 Forward primer씤 16S-rRNA1 (5’-AGT TTG ATC CTG GCT CAG-3’) Reverse primer씤 16S-rRNA2 (5’-GGT TAC CTT GTT ACG ACT T-3’)瑜 씠슜븯떎. Primer 媛곴컖 20 pmol 1 μL, dNTPs 1 μL, 10x Reaction buffer (1x: 10 mM Tris-HCL, 1.5 mM KCL, 0.1% Triton X-100) 5 μL, 2.5 U/μL DyNAzyme™ polymerase 1 μL, 硫멸퇏 DW 38.5 μL뿉 異붿텧맂 gDNA 2.5 μL瑜 꽔뼱 珥 50 μL濡 諛섏쓳븸쓣 留뚮뱾뿀떎. PCR TaKaRa PCR Thermal cycler (TP600 Gradient, Roche Molecular System, CA, USA)瑜 궗슜븯怨, 삩룄 議곌굔쓣 94°C뿉꽌 5遺 predenaturation떆궓 썑, 94°C뿉꽌 1遺 denaturation, 49°C뿉꽌 1遺 annealing, 72°C뿉꽌 1遺 extension쓣 1二쇨린濡 36쉶 떎떆븯뿬 利앺룺븯怨 72°C뿉꽌 10遺꾧컙 postextension쓣 떎떆븯떎.

PCR 利앺룺궛臾쇱 1.8% agarose gel쓣 궗슜븯뿬 100 V濡 30遺꾧컙 loading떆耳 UV 닾怨쇱“紐낃린뿉꽌 솗씤븯怨, Gene-All PCR DNA Purification kit (Geneall Biotechnology Co. LTD Seoul, Korea)瑜 궗슜븯뿬 PCR 궛臾쇱쓣 젙젣븯떎. 洹 썑 Big dye PCR쓣 떆뻾븯쑝硫 Big dye PCR쓽 寃쎌슦 Seq primer瑜 씠슜븯떎. Forward reverse primer瑜 씠슜빐 媛곴컖 떆뻾븯怨, 諛섏쓳븸 5 pmol seq primer 2 μL, 젙젣맂 PCR 利앺룺궛臾 3 μL, Big-dye 0.5 μL, PCR buffer 3 μL, 硫멸퇏 DW 4.5 μL瑜 샎빀븯뿬 珥 13 μL媛 릺룄濡 븳 썑 諛섏쓳쓣 吏꾪뻾븯떎. 96°C뿉꽌 1遺꾧컙 predenaturation쓣 닔뻾븯怨, 96°C 10珥, 50°C 5珥, 60°C 4遺꾩쑝濡 援ъ꽦맂 닚솚議곌굔쓣 25쉶 諛섏쓳떆궓 썑 3 M sodium acetate瑜 씠슜븳 移⑥쟾怨쇱젙怨 95% EtOH瑜 씠슜븳 깉닔怨쇱젙쓣 嫄곗퀜 諛섏쓳臾쇱쭏留뚯쓣 젙젣븯떎. 젙젣맂 諛섏쓳臾쇱 嫄댁“떆耳 Hidi 10 μL뿉 끃씤 썑 ABI 3730XL DNA Analyzer (Applied Biosystems)瑜 씠슜빐 뿼湲곗꽌뿴쓣 遺꾩꽍븯떎. 遺꾩꽍븳 뿼湲곗꽌뿴쓽 쑀궗꽦 寃깋 NCBI data base씤 BLAST瑜 씠슜븯떎. 遺꾩꽍븳 옄猷 以 A≥99.0%쓽 쑀궗꽦쓣 蹂댁씠뒗 寃곌낵瑜 CLSI쓽 湲곗쓣 쟻슜븯뿬 뙋룆븯떎(Chun et al., 2007; Wayne, 2007).

寃곌낵

遺꾧린蹂 삁븸 諛곗뼇뿉꽌 洹몃엺 뼇꽦 留됰洹(GPB)쓽 遺꾨━ 鍮덈룄

2014뀈 10썡遺꽣 2016뀈 9썡源뚯 2뀈媛 삁븸 諛곗뼇씠 쓽猶곕맂 珥 87,241嫄 以 GPB쓽 遺꾨━ 鍮꾩쑉 2014뀈 4遺꾧린 83嫄(0.8%), 2015뀈 1遺꾧린 68嫄(0.7%), 2015뀈 2遺꾧린 83嫄(0.9%), 2015뀈 3遺꾧린 124嫄(1.1%), 2015뀈 4遺꾧린 84嫄(0.8%), 2016뀈 1遺꾧린 65嫄(0.6%), 2016뀈 2遺꾧린 105嫄(0.9%), 2016뀈 3遺꾧린 101嫄(0.8%)씠뿀떎. 뿰援ш린媛 룞븞 삁븸 諛곗뼇씠 쓽猶곕맂 쟾泥 嫄댁닔 以 삁븸 諛곗뼇 뼇꽦瑜좎 10.0%쑝硫, GPB 뼇꽦瑜좎 0.8%떎(Table 1).

Isolation rate of Gram positive bacilli in blood culture (2014-2016)

Quarter of a year4th quarter of 2014 N (%)1st quarter of 2015 N (%)2nd quarter of 2015 N (%)3rd quarter of 2015 N (%)4th quarter of 2015 N (%)1st quarter of 2016 N (%)2nd quarter of 2016 N (%)3rd quarter of 2016 N (%)Total
No. (%) of cases83/ 10168 (0.8%)68/ 9732 (0.7%)83/ 9574 (0.9%)124/ 11255 (1.1%)84/ 11163 (0.8%)65/ 11321 (0.6%)105/ 11937 (0.9%)101/ 12091 (0.8%)713/ 87241 (0.8%)

1st quarter: Jan to Mar, 2nd quarter: Apr to Jun, 3rd quarter: Jul to Sep, 4th quarter: Oct to Dec


MALDI-TOF MS System쓣 씠슜븳 삁븸 諛곗뼇쑝濡쒕꽣 遺꾨━맂 洹몃엺 뼇꽦 留됰洹(GPB)쓽 룞젙

1.7~2.0쓽 cut-off score瑜 굹궦 GPB쓽 遺꾨━ 鍮꾩쑉: Cut-off score 1.7~20쓣 굹궦 GPB 以 Bacillus subtilis濡 룞젙맂 鍮꾩쑉 19嫄(14.1%), Bacillus cereus뒗 15嫄(11.9%), Corynebacterium striatum뒗 14嫄(10.4%), Corynebacterium afermentans뒗 10嫄(7.4%), Bacillus mojavensis뒗 8嫄(5.9%)씠뿀떎(Table 2).

Identification rate of Gram positive bacilli which showed 1.7 to 2.0 of MALDI-TOF MS cut-off scores (2014-2016)

 Species identification results by MALDI-TOF MS systemNo. of isolates with the number of positive blood cultures per patientTotal No. (%) of patientTotal No. (%) of species

12345
Bacillus subtilis19000019 (15.6)19 (14.1)
Bacillus cereus14100015 (12.3)16 (11.9)
Corynebacterium striatum410207 (5.7)14 (10.4)
Corynebacterium afermentans10000010 (8.2)10 (7.4)
Bacillus mojavensis800008 (6.6)8 (5.9)
Bacillus pumilus800008 (6.6)8 (5.9)
Bacillus sonorensis700007 (5.7)7 (5.2)
Bacillus licheniformis600006 (4.9)6 (4.4)
Bacillus vallismortis500005 (4.1)5 (3.7)
Mycobacterium abscessus000101 (0.8)4 (3.0)
Bacillus megaterium400004 (3.3)4 (3.0)
Microbacterium species300003 (2.5)3 (2.2)
Clostridium perfringens110002 (1.6)3 (2.2)
Eggerthella lenta200002 (1.6)2 (1.5)
Brevibacterium iodium010001 (0.8)2 (1.5)
Actinomyces odontolyticus200002 (1.6)2 (1.5)
Paenibacillus urinalis200002 (1.6)2 (1.5)
Solibacillus silvestis200002 (1.6)2 (1.5)
Bacillus circulans and othersa18000018 (14.8)18 (13.3)

  Total1154030122 (100.0)135 (100.0)

aBacillus muralis, Bacillus mycoisea, Bacillus niacini, Bacillus thuringiensis, Bacillus weienstephanensis, Clostridium bifermentans, Clostridium carnis, Clostridium innocuum, Corynebacterium singulare, Corynebacterium tuscaniense, Dermabacter hominis, Gordonarubropertincta, Lysinibacillus fusiformis, Microbacterium oxydans, Microbacterium testaceum, Paenibacillus latus, Paenibaillus xylanilyticus.


2.0 씠긽쓽 cut-off score瑜 굹궦 洹몃엺 뼇꽦 留됰洹(GPB)쓽 遺꾨━ 鍮꾩쑉: Cut-off score瑜 1.7 씠긽 굹궡硫, GPB濡 룞젙맂 珥 410嫄 以 275嫄(67.1%) 2.0 씠긽쓽 넂 cut-off score濡 GPB쓽 醫 룞젙씠 媛뒫븯떎. Cut-off score 2.0 씠긽쓣 굹궦 GPB 以 C. striatum 66嫄(27.3%), B. cereus뒗 44嫄(16.0%), Paenibacillus urinalis 27嫄(9.8%), Listeria monocytogenes뒗 25嫄(9.1%), Clostridium perfringens뒗 15嫄(5.5%)씠뿀떎(Table 3).

Identification rate of Gram positive which showed above 2.0 of MALDI-TOF MS cut-off scores (2014-2016)

 Species identification results by MALDI-TOF MS systemNo. of isolates with the number of positive blood cultures per patientTotal No. (%) of patientTotal No. (%) of species

12345678
Corynebacterium striatum17913201134 (16.7)75 (27.3)
Bacillus cereus35310000039 (19.1)44 (16.0)
Paenibacillus urinalis25100000026 (12.7)27 (9.8)
Listeria monocytogenes3702000012 (5.9)25 (9.1)
Clostridium perfringens9300000012 (5.9)15 (5.5)
Bacillus subtilis11000000011 (5.4)11 (4.0)
Bacillus licheniformis10000000010 (4.9)10 (3.6)
Clostridium tertium600000006 (2.9)6 (2.2)
Microbacterium species100100002 (1.0)5 (1.8)
Actinomyces oris400000004 (2.0)5 (1.8)
Bacillus infantis400000004 (2.0)4 (1.5)
Bacillus pumilus400000004 (2.0)4 (1.5)
Bacillus megaterium300000003 (1.5)3 (1.1)
Bacillus sonorensis300000003 (1.5)3 (1.1)
Clostridium carnis001000001 (0.5)3 (1.1)
Eggerthella lenta300000003 (1.5)3 (1.1)
Bacillus mojavensis200000002 (1.0)2 (0.7)
Bacillus mycoisea200000002 (1.0)2 (0.7)
Corynebacterium minutissimun200000002 (1.0)2 (0.7)
Corynebacterium urealyticum010000001 (0.5)2 (0.7)
Lactobacillus paracasei200000002 (1.0)2 (0.7)
Microbacterium aurum200000002 (1.0)2 (0.7)
Mycobacterium abscessus010000001 (0.5)2 (0.7)
Paenibacillus illinoisensis200000002 (1.0)2 (0.7)
Actinomyces odontolyticus and othersa16000000016 (7.8)16 (5.8)

   Total16625362011204 (100.0)275 (100.0)

aArthrobacteroxydans, Bacillus atrophaeus, Bacillus flexus, Bacillus thermoamylovorans, Bacillus thuringiensis, Corynebacterium amycolatum, Corynebacterium falsenii, Corynebacterium pseudodiphthriticum, Exigubacterium aurantiacum, Lactobaillus salivarius, Lysinibacillus fusiformis, Paenibacillus barengoltzii, Paenibacillus rhizosphaerae, Paenibacillus odorifer, Paenibacillus pauli.


MALDI-TOF MS 遺꾩꽍 cut-off score뿉 뵲瑜 洹몃엺 뼇꽦 留됰洹(GPB)쓽 遺꾨━ 鍮꾩쑉:C. striatumB. cereus뒗 媛곴컖 2뀈 룞븞 遺꾨━맂 珥 89嫄 以 66嫄(74.2%), 60嫄 以 44嫄(73.3%)씠 cut-off score 2.0 씠긽쓽 넂 닔移섎줈 룞젙 媛뒫븯怨, L. monocytogenes C. perfringens뒗 媛곴컖 25嫄 以 25嫄(100%), 18嫄 以 15嫄(83.3%)씠 cut-off score 2.0 씠긽쓽 넂 닔移섎줈 룞젙 媛뒫븯떎(Table 4).

Distribution of Gram positive bacilli species from blood culture according to the cut-off scores of MALDI-TOF MS (2014-2016)

 Species identification results by MALDI-TOF MS systemScoreScoreTotal No. of species
≤ 2.0 ~ ≥ 1.7≥ 2.0
Corynebacterium striatum147589
Bacillus cereus164460
Bacillus subtilis191130
Paenibacillus urinalis22729
Listeria monocytogenes02525
Clostridium perfringens31518
Bacillus licheniformis61016
Bacillus pumilus8412
Other species6764131
Not reliable identification--213
No peaks found--90

  Total135275713

삁븸 諛곗뼇쑝濡쒕꽣 遺꾨━맂 洹몃엺 뼇꽦 留됰洹(GPB) 룞젙쓣 쐞븳 16S rDNA 뿼湲곗꽌뿴 遺꾩꽍怨 MALDI-TOF MS system쓽 寃곌낵 鍮꾧탳

꽭洹좎쓽 16S rDNA 뿼湲곗꽌뿴 遺꾩꽍怨 MALDI-TOF MS 遺꾩꽍쓣 룞떆뿉 떎떆븳 珥 50嫄 以 43嫄(86.0%) 몢 遺꾩꽍뿉꽌 寃곌낵媛 씪移섑븯쑝硫, 7嫄(14.0%) 寃곌낵媛 씪移섑븯吏 븡븯떎. 몢 遺꾩꽍뿉꽌 씪移섑븳 寃곌낵瑜 굹궦 寃쎌슦뒗 C. striatum 19嫄, L. monocytogenes 9嫄, C. perfringens 5嫄, B. iodinum 1嫄, Clostridium canis 1嫄, Corynebacterium urealyticum 1嫄, Mycobacterium abscessus 1嫄, P. urinalis 1嫄 씠뿀떎. 몢 遺꾩꽍뿉꽌 遺덉씪移섎 굹궦 7嫄 以 MALDI-TOF MS 遺꾩꽍뿉꽌 cut-off score 1.7 씠긽쓣 굹깉뜕 寃쎌슦뒗 珥 2嫄댁쑝濡 B. cereusLysinbacillus spp.濡, Microbacterium spp.뒗 Mycobacterium paraoxydans濡 媛곴컖 遺덉씪移섑븳 寃곌낵瑜 굹깉떎. 몢 遺꾩꽍뿉꽌 遺덉씪移섑븳 寃곌낵瑜 굹궦 굹癒몄 5二쇰뒗 MALDI-TOF MS 遺꾩꽍뿉꽌 not reliable identification 샊 no peaks found쓽 寃곌낵瑜 굹깉怨, 16S rDNA 遺꾩꽍쓣 떎떆븳 寃곌낵 媛곴컖 Cellulomonas hominis, Corynebacterium amycdatum, Catabacter hongkongenesis, Bacillus spp., Bacillus velezensis濡 룞젙릺뿀떎(Table 5).

Comparison of the results of MALDI-TOF MS and 16S rDNA sequence analysis with double positive cases in blood culture of the same patient

 Species identification results by MALDI-TOF MS SystemNo. of isolatesFianl ID result by 16S-rRNA sequencing

2.0 ≤ Score1.7 ≤ Score ≤ 2.0Score < 1.7Concordant resultDiscordant result
Corynebacterium striatum172190
Bacillus cereus5151Lysinbacillus sp.
Clostridium perfringens4150
Listeria monocytogenes990
Bacillus iodinum110
Clostridium carnis110
Corynebacterium urealyticum110
Microbacterium species101Mycobacterium paraoxydans
Mycobacterium abscessus110
Paenibacillus urinalis110
Not reliable identification404Cellulomonas hominis
Corynebacterium amycolatum
Catabacter hongkongenesis
Bacillus sp.
No peaks found01Bacillus velezensis

  Total3964437

怨좎같

湲곗〈쓽 뿰援щ뱾쓣 넻빐 엫긽쟻쑝濡 쓽쓽媛 엳뒗 GPC, GNB, 吏꾧퇏쓽 遺遺꾩 MALDI-TOF MS 遺꾩꽍쑝濡 醫낃낵 냽쓽 닔以源뚯 媛꾪렪븯怨 떊냽븯寃 룞젙씠 媛뒫븿씠 쁽옱源뚯 솗씤릺뿀떎(Bernard et al., 2012; Levesque et al., 2015). 븯吏留, 긽쟻쑝濡 삤뿼洹좎쑝濡 뿬寃⑥뜕 GPB뿉 븳 湲곗〈쓽 뿰援ъ 엫긽쟻 쑀슜꽦 룊媛 寃곌낵뒗 寃곗뿬릺뼱 엳뼱 蹂 뿰援щ 넻빐 삁븸 諛곗뼇뿉꽌 遺꾨━릺뒗 GPB쓽 룞젙씠 MALDI-TOF MS 遺꾩꽍쓣 넻빐 쑀슜꽦씠 엳뒗吏 遺꾩꽍빐 蹂댁븯떎. 뵲씪꽌, 援궡쓽 븳 븰蹂묒썝뿉꽌 2뀈媛 떆뻾맂 珥 87,241嫄댁쓽 삁븸 諛곗뼇 以 GPB媛 遺꾨━맂 713嫄(0.8%)쓣 긽쑝濡 뿰援щ 떎떆븯떎.

옣鍮 쉶궗뿉꽌 젣怨듯븯뒗 湲곗뿉 뵲씪 洹좉낵 냽쓽 룞젙씠 媛뒫븳 cut-off score 1.7 씠긽쓣 굹궦 寃쎌슦뒗 珥 410嫄(57.5%)씠뿀떎. 룞젙 媛뒫븯쑝굹, cut-off score媛 1.7 씠븯濡 떊猶고븷 닔 뾾뒗 寃곌낵瑜 蹂댁뜕 寃쎌슦뒗 珥 213嫄(29.9%) 씠뿀쑝硫, 遺꾩꽍 寃곌낵媛 No peaks found濡 룞젙議곗감 옒뱾뿀뜕 寃쎌슦뒗 90嫄(12.6%)뿉 떖뻽떎. Cut-off score 1.7 씠긽쓣 굹궦 寃쎌슦 C. striatum쑝濡 룞젙맂 寃쎌슦뒗 89嫄(12.4%), B. cereus뒗 60嫄(8.4%), B. subtilis 30嫄(4.2%), P. urinalis뒗 29嫄(4.1%), L. monocytogenes뒗 25嫄(3.5%)씠뿀떎. Corynebacterium spp., Bacillus spp., Listeria spp. 엫긽쟻쑝濡쒕룄 쓽쓽媛 留롮 媛먯뿼洹좎쑝濡쒖꽌 넂 鍮꾩쑉쓣 蹂댁엫쓣 븣 닔 엳뿀떎(Renom et al., 2012; Severo et al., 2014). 삉븳 遺꾨━ 鍮꾩쑉 쟻吏留 Mycobacterium spp.怨 Clostridium spp. cut-off score 1.7 씠긽쓽 떊猶고븷 닔 엳뒗 寃곌낵濡 룞젙씠 媛뒫븿쓣 븣 닔 엳뿀떎. 臾쇰줎 not reliable identification 샊 no peaks found쓽 寃곌낵瑜 굹궦 寃쎌슦룄 42.5%濡 넂 鍮꾩쑉쓣 李⑥븯쑝굹, 嫄곗쓽 遺遺꾩쓽 寃쎌슦 1쉶 遺꾨━洹좎 삤뿼洹좎쑝濡 媛꾩<릺湲 븣臾몄뿉 媛먯뿼洹좎쑝濡쒖쓽 쓽쓽媛 뾾뒗 寃껋쑝濡 궗猷뚮맂떎.

Cut-off score 2.0 씠긽쑝濡 룞젙맂 洹 醫낃낵 cut-off score 1.7~2.0쑝濡 룞젙맂 洹 醫낆쓣 鍮꾧탳빐 蹂대㈃ cut-off score 1.7 씠긽쑝濡 룞젙맂 珥 410嫄 以 275嫄(67.1%) 2.0 씠긽쓽 넂 cut-off score濡 룞젙씠 媛뒫뻽떎. Cut-off score 2.0 씠긽씤 寃쎌슦뒗 C. striatum씠 66嫄(27.3%), B. cereus媛 44嫄(16.0%), P. urinalis媛 27嫄(9.8%), L. monocytogenes媛 25嫄(9.1%), C. perfringens媛 15嫄(5.5%)씠뿀怨, cut-off score 1.7~2.0濡 룞젙맂 洹 醫낆 B. subtilis媛 19嫄(14.1%), B. cereus媛 15嫄(11.9%), C. striatum媛 14嫄(10.4%), C. afermentans媛 10嫄(7.4%), B. mojavensis媛 8嫄(5.9%)씠뿀떎. C. striatumB. cereus뒗 媛곴컖 2뀈 룞븞 遺꾨━맂 珥 89嫄 以 66嫄(74.2%), 60嫄 以 44嫄(73.3%)쓣 cut-off score 2.0 씠긽쓽 넂 떊猶곕룄瑜 媛吏怨 룞젙씠 媛뒫뻽떎. 삉븳, 1쉶留 遺꾨━릺뼱룄 쓽쓽媛 엳뒗 L. monocytogenes뒗 25嫄 紐⑤몢媛 cut-off score 2.0 씠긽쓣 굹깉떎.

GPB씠吏留 룞씪 솚옄뿉꽌 2쉶 씠긽 遺꾨━맂 寃쎌슦 媛먯뿼썝쑝濡 媛꾩<릺湲 븣臾몄뿉 씠윭븳 議곌굔뿉 빐떦븯뒗 珥 50嫄댁 16S rDNA 뿼湲곗꽌뿴 遺꾩꽍쓣 넻빐 옱솗씤 젅李⑤ 吏꾪뻾븯쑝硫, 理쒖쥌 寃곌낵瑜 鍮꾧탳빐 蹂댁븯떎. 珥 50嫄 以 43嫄(86.0%) MALDI-TOF MS 16S rDNA 뿼湲곗꽌뿴 遺꾩꽍 寃곌낵媛 씪移섑븯怨, 굹癒몄 7嫄(14.0%) 遺덉씪移 븯뒗 寃곌낵瑜 굹깉떎.

蹂 뿰援ъ쓽 寃곌낵瑜 넗濡 吏궃 2뀈媛꾩쓽 삁븸 諛곗뼇뿉꽌 遺꾨━맂 GPB쓽 젅諛 씠긽씠 MALDI-TOF MS 遺꾩꽍 떆 cutoff score 1.7 씠긽쓽 떊猶고븷 닔 엳쓣留뚰븳 닔以쑝濡 洹 醫낅룞젙씠 媛뒫뻽怨, 洹 以 1쉶留 遺꾨━릺뼱룄 엫긽쟻 쓽쓽媛 엳뒗 洹 醫, 룞씪 솚옄뿉꽌 2쉶 씠긽 遺꾨━릺뼱 삤뿼洹좎씠 븘땶 媛먯뿼洹좎쑝濡 媛꾩<릺뒗 洹 醫낆 꽭洹 16S rDNA 뿼湲곗꽌뿴 遺꾩꽍 寃곌낵 86.0%쓽 넂 씪移섏쑉쓣 굹깉떎. 異붽쟻쑝濡, 꽭洹좎쓽 16S rDNA 뿼湲곗꽌뿴 遺꾩꽍怨 MALDI-TOF MS 遺꾩꽍쓣 룞떆뿉 떎떆븳 珥 50嫄 以 39嫄(78.0%) cutoff score 2.0 씠긽, 6嫄(12.0%) cut-off score 1.7 씠긽쓽 넂 떊猶곕룄濡 洹 醫 룞젙씠 媛뒫뻽쑝硫, 珥 2嫄(4.0%)뿉꽌留 MALDI-TOF MS 꽭洹좎쓽 16S rDNA 뿼湲곗꽌뿴 遺꾩꽍 寃곌낵媛 씪移섑븯吏 븡븯떎. 씠윭븳 寃곌낵뱾쓣 湲곕컲쑝濡 븯뿬, 湲곗〈뿉 삤뿼洹좎쑝濡 깮媛곷릺뼱 嫄곗쓽 룞젙릺吏 븡븯뜕 GPB洹 醫낆쓣 MALDI-TOF MS System쓣 씠슜빐 1李 룞젙쓣 떎떆븯怨, 엫긽쟻 쓽쓽媛 엳뒗 洹 醫낆뿉 빐꽌뒗 16S rDNA 뿼湲곗꽌뿴 遺꾩꽍쓣 異붽쟻쑝濡 쟻슜븯뿬 寃곌낵瑜 솢슜븳떎硫, 뼢썑 媛먯뿼썝쑝濡쒖꽌 쓽쓽媛 엳뒗 GPB 룞젙뿉 留ㅼ슦 쑀슜븷 寃껋쑝濡 궗猷뚮맂떎.

븯吏留, 솚옄濡쒕꽣 쓷엳 遺꾨━릺吏 븡怨, 鍮꾨퀝썝꽦 꽭洹좎씠 留롮씠 룷븿릺뼱 엳뒗 GPB쓽 듅꽦쑝濡 씤빐 쁽옱 MALDI-TOF MS瑜 씠슜빐 洹 醫 룞젙쓣 븯湲 쐞븳 뜲씠꽣踰좎씠뒪媛 遺議깊븯怨, 삊留됱씠 議댁옱븯뒗 씪遺 GPB쓽 援ъ“쟻 듅꽦긽 떒諛깆쭏 遺꾨━쓽 뼱젮씠 엳湲 븣臾몄뿉 룞젙씠 옒뱺 寃쎌슦媛 留롮씠 솗씤릺뿀떎. 異뷀썑 MALDI-TOF MS瑜 씠슜빐 GPB 洹 醫낆쓣 룞젙븷 닔 엳뒗 뜲씠꽣踰좎씠뒪瑜 뜑슧 솗異⑺븯怨, 삊留 蹂댁쑀 꽭洹좎쓣 쐞빐 異붽 蹂댁셿맂 떒諛깆쭏 遺꾨━ 諛⑸쾿씠 궗슜맂떎硫, GPB쓽 떊냽 젙솗븳 룞젙뿉 留롮 룄씠 맆 닔 엳쓣 寃껋쑝濡 궗猷뚮맂떎.

ACKNOWLEDGEMENTS

None.

CONFLICT OF INTEREST

The authors have no conflicts of interest to disclose.

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