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諛곗醫낅쪟 諛곗뼇떆媛꾩뿉 뵲瑜 移대컮럹꽩궡꽦 옣궡꽭洹좎쓽 MALDI Biotyper 룞젙뒫 룊媛
Performance of MALDI Biotyper for Species Identification of Carbapenem-Resistant Enterobacteriaceae by Media Types and Incubation Time
J Lab Med Qual Assur 2018;40:155-160
Published online September 30, 2018
© 2018 Korean Association of External Quality Assessment Service.

議곗쁺, 理쒕퀝썑, 옣젙쁽, 꽦씎꽠, 源誘몃굹
Young Eun Cho, Byoung Hu Choi, Jeonghyun Chang, Heungsup Sung, Mi-Na Kim

슱궛븰援 쓽怨쇰븰 꽌슱븘궛蹂묒썝 吏꾨떒寃궗쓽븰怨

Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
Correspondence to: Mi-Na Kim Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea Tel: +82-2-3010-4511 Fax: +82-2-478-0884 E-mail: mnkim@amc.seoul.kr
援먯떊옄: 源誘몃굹 슦)05505 꽌슱떆 넚뙆援 삱由쇳뵿濡43湲 88, 슱궛븰援 쓽怨쇰븰 꽌슱븘궛蹂묒썝 吏꾨떒寃궗쓽븰怨 Tel: 02)3010-4511, Fax: 02)478-0884, E-mail: mnkim@amc.seoul.kr
Received January 3, 2018; Revised July 10, 2018; Accepted August 2, 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

諛곌꼍:

T移대컮럹꽩궡꽦 옣궡꽭洹(carbapenem-resistant Enterobacteriaceae, CRE)쓽 媛먯떆諛곗뼇諛곗뿉꽌 옄 吏묐씫쓣 吏곸젒 matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF)瑜 씠슜븯뿬 룞젙븷 븣 諛곗醫낅쪟 諛곗뼇떆媛꾩뿉 뵲瑜 룞젙뒫쓣 룊媛븯떎.

諛⑸쾿:

2017뀈 3썡遺꽣 2떖媛 쓽猶곕맂 CRE 媛먯떆諛곗뼇뿉꽌 imipenem씠 1 쨉g/mL 泥④맂 MacConkey agar (IMP-Mac)뿉꽌 뼇꽦쑝濡 꽑蹂꾨맂 吏묐씫뱾쓣 MALDI Biotyper (Bruker Daltonics, Germany)瑜 씠슜븯뿬 吏곸젒 룞젙븯떎. 삉븳 삁븸븳泥쒕같吏(blood agar, BA), Mueller Hinton agar (MH), MacConkey agar (Mac)뿉 怨꾨諛곗뼇븯뿬 媛곴컖쓽 諛곗뿉 븯猷삳갇 諛곗뼇븳 寃껉낵 씠吏멸퉴吏 諛곗뼇븳 寃껋쓣 鍮꾧탳룊媛븯떎. 냽닔以源뚯 룞젙뿉 꽦怨듯븳 寃쎌슦 룞젙瑜좉낵 룞젙븳 뒪肄붿뼱 媛믪쓣 遺꾩꽍븯떎.

寃곌낵:

珥 117媛 寃泥댁뿉꽌, Klebsiella pneumoniae 84媛, Escherichia coli 12媛, Enterobacter cloacae 9媛, Klebsiella oxytoca 5媛, Enterobacter aerogenes 4媛, Raoultella ornithinolytica 2媛쒓 룞젙릺뿀떎. Mac IMP-Mac쓽 룞젙瑜좎씠 82.1% (P<0.001) 70.9% (P<0.001)뜕 諛섎㈃, BA MH쓽 룞젙瑜좎 媛곴컖 98.3%, 97.4% (P=0.9)떎. 諛곗뼇 씠吏 BA, MH, Mac怨 IMP-Mac 諛곗蹂 룞젙瑜좎 96.6%, 96.6% (P=1.0), 61.5% (P<0.001), 58.1% (P<0.001)濡 Mac怨 IMP-Mac뿉꽌 쑀쓽븯寃 媛먯냼븯떎. MALDI 뒪肄붿뼱 룊洹좉컪 Mac (2.017짹0.22)怨 IMP-Mac (1.978짹0.24)씠 BA (2.213짹0.16) (P<0.001)뿉 鍮꾪빐 쑀쓽븯寃 궙븯떎.

寃곕줎:

Mac 삉뒗 IMP-Mac뿉꽌 吏곸젒 MALDI Biotyper濡 룞젙븯뒗 寃쎌슦 룞젙瑜좎씠 궙븯떎. 씠뒗 MALDI-TOF 룞젙 쟾 BA 삉뒗 MH뿉꽌 븯猷삳갇 怨꾨諛곗뼇븿쑝濡쒖뜥 룞젙 젙솗룄瑜 넂씪 닔 엳떎뒗 寃껋쓣 굹궦떎.

Background:

This study was conducted to evaluate the impact of the media type used for direct identification of colonies on the surveillance culture of carbapenem-resistant Enterobacteriaceae (CRE) by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Methods:

CRE surveillance culture isolates were subjected to species identification using the MALDI Biotyper (Bruker Daltonics, Germany) for 2 months starting in March 2017. Four types of media were evaluated: blood agar (BA), Mueller Hinton agar (MH), MacConkey agar (Mac), and MacConkey agar containing imipenem of 1 쨉g/mL (IMP-Mac). CRE-like colonies on IMP-Mac and their subculture colonies on the other media were tested after overnight incubation and extended incubation for one additional day. The percent identification and score value were analyzed for each media types and incubation time when the identification was correct at the genus level.

Results:

A total of 117 isolates were identified as 84 Klebsiella pneumoniae, 12 Escherichia coli, 9 Enterobacter cloacae, 5 Klebsiella oxytoca, 4 Enterobacter aerogenes, and 2 Raoultella ornithinolytica. The successful identification rates (SIR) for BA and MH were 98.3% and 97.4% (P=0.9), respectively, while those for Mac and IMP-Mac were 82.1% (P<0.001) and 70.9% (P<0.001), respectively. After extended incubation, SIRs were decreased to 96.6%, 96.6% (P=1.0), 61.5% (P<0.001), and 58.1% (P<0.001) on BA, MH, Mac, and IMP-Mac, respectively. The average score values were significantly lower for Mac (2.017짹0.22) and IMP-Mac (1.978짹0.24) than for BA (2.213짹0.16) (P<0.001).

Conclusions:

The low performance of the MALDI Biotyper applied directly to the colonies grown on Mac or IMP-Mac indicates that subculture on BA or MH is preferable before identification by MALDI-TOF MS.

Keywords : Carbapenem-resistant Enterobacteriaceae, Surveillance, MALDI-TOF, Media type, Incubation time
INTRODUCTION

Carbapenem-resistant Enterobacteriaceae (CRE) represent a serious growing threat to public health in Korea. Serious Infections with CRE are difficult to treat or non-treatable, and thus are associated with high mortality rates.왉arbapenemase-mediated CRE is unique because of its capacity for horizontal transmission via a plasmid, by which carbapenem-resistance increases rapidly and crosses the species barrier [1]. In a previous윹tudy of five Korean university hospitals, ertapenem susceptibilities were 99.5% among Escherichia coli and 94.0% among Klebsiella pneumoniae in 2015 [2]. CRE has been designated as a legally notifiable disease since October 2010, and Korea Centers for Disease Control and Prevention (KCDC) implemented a laboratory-based monitoring system in representative regional hospitals to collect and genotype CRE and CPE (Carbapenemase-producing Enterobacteriaceae) nationwide [1]. CRE has been considered as a group III notifiable disease since June 2017, and all CRE cases and patients harboring CRE should be reported to the KCDC [1].

CRE surveillance culture is an essential element in the control and prevention of CRE infection [3]. Because CRE infection rates have increased since the first four cases of NDM-producing K. pneumoniae were found at our hospital in 2010 [4], CRE surveillance culture has become a routine practice in many clinical microbiology laboratories in Korea. To accelerate the turn-around time of CRE surveillance culture, matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS, MALDI-TOF) was recently introduced for direct species identification of colonies on selective agar [5-7]. However, species identification of CRE grown on MacConkey agar containing imipenem (IMP-Mac) failed biochemical identification tests in this hospital.

This study was performed to evaluate the impact of the media type on species identification using MALDI-TOF in CRE surveillance cultures. The influence of colony aging on species identification was also measured for incubation times of 1 and 2 days.

MATERIALS AND METHODS

1. Specimens

For 2 months, March and April 2017, four types of media were evaluated: blood agar (BA; KoMed, Seongnam, Korea), Mueller Hinton agar agar (MH, KoMed), MacConkey agar (Mac, KoMed), MacConkey agar containing imipenem of 1 關g/mL (IMP-Mac). The samples subjected to CRE surveillance culture were inoculated on IMP-Mac and pink colonies were considered as CRE-likely colonies after overnight incubation at 36째C짹1째C. Because routine practices in the laboratories use extended culture at room temperature to detect colonies in the case of holiday, the incubation was extended for one additional day at room temperature. All pink colonies on IMP-Mac were consecutively collected and subcultured on BA and Mac plates. Disk diffusion tests were conducted on MH agar. The colonies on each media type were tested for species identification using a MALDI biotyper (Bruker Daltonics, Bremen, Germany) and MicroScan NC72 panel (Beckman Coulter, Brea, CA, USA).

2. MALDI-TOF Identification

The colonies on each agar plate were directly prepared for MALDI-TOF identification. Briefly, a single colony was picked using a wooden toothpick and gently smeared onto a polished steel target plate. The spots were then overlaid with 1 關L of IVD matrix solution (Bruker Daltonics) consisting of 47.5% deionized water, 50% acetonitrile (Sigma-Aldrich, St. Louis, MO, USA) and 2.5% trifluoroacetic acid (Sigma-Aldrich) and allowed to dry. The MALDI biotyper operation was conducted according to the manufacturer셲 instructions and species identification was based on Bruker Taxonomy with Mass Spectrometry Profiling. According to the manufacturer셲 instructions, the identification results with score values 돟1.7 were considered reliable at the genus level. The correct identification was considered as showing consistent with results with MicroScan NC72 or Klebsiella oxytoca in MicroScan NC72 for Raoultella ornithinolytica identified by Biotyper. The successful identification rates (SIRs) and score values of the identification results were analyzed for each media type and incubation time.

3. Statistical Analysis

The SIRs of each media type were compared by chi-square tests. The score values of each specimen resulting in correct identifications were compared by paired t-test. Analysis was conducted using PASW SPSS ver. 18.0 software (SPSS Inc., Chicago, IL, USA). P-value of <0.05 was considered statistically significant.

RESULTS

A total of 117 CRE were detected, including 84 K. pneumoniae, 12 Escherichia coli, 9 Enterobacter cloacae, 5 K. oxytoca, 4 Klebsiella aerogenes, 2 R. ornithinolytica, and 1 Citrobacter freundii. The MALDI-TOF identification results are shown in Table 1. At overnight incubation, 98.3% of isolates were correctly identified from the colonies on BA, 97.4% on MH (P=0.9), 82.1% (P<0.001) on MAC, and 70.9% (P<0.001) on IMP-MAC. After 2 days of incubation, the SIRs were decreased to 61.5% (P<0.001) on MAC and 58.1% (P<0.001) on IMP-MAC, but not on BA and on MH which showed values of 96.6% and 96.6%, respectively. Unsuccessful identification mainly resulted from the lack of a peak, and the number of samples without peaks was increased after 2 days of incubation: 2 to 4 in BA, 3 to 4 in MH, 16 to 37 in Mac, and 24 to 37 in IMP-Mac (Table 1). The average score values for correctly identified specimens were significantly lower for Mac (2.017짹0.22, P<0.001) and IMP-Mac (1.978짹0.24, P<0.001), while there was no significant difference between those for MH (2.300짹0.14) and BA (2.213짹0.16). After 2-day incubation, the score values were slightly lower for each media type than those after day 1, but the differences were not significant among the specimens identified correctly except for E. coli on Mac and E. cloacae on IMP-Mac (Table 1). Among species for which 4 or more isolates were examined in this study, identification performance of the Biotyper was significantly lower when colonies grown on Mac or IMP-Mac were used, except for E. cloacae. The SIRs of colonies grown on Mac were further decreased by colony aging, except for E. coli, while those grown on BA and MH were not (Table 1).

Table 1 . Species identification and score value of carbapenem-resistant Enterobacteriaceae using Biotyper by media types and incubation times.

꺿긕peciesNo. of isolateIncubation time (day)No. of successful identification (%)/no. of no peak (%), score value of correct identification, mean짹SD (no. of measurements)

꺿꺿귻lood agar꺿꺿긎uller Hinton Agar꺿꺿긎acConkey Agar꺿꺿긆MP-MacConkey agar
Klebsiellap pneumoniae84182 (97.6)/2 (2.4), 2.209짹0.16 (82)82 (97.6)/2 (2.4), 2.258짹0.14 (82) P=1.0/P=0.0268 (81.0)/12 (14.3), 2.006짹0.23 (72) P<0.001/P<0.00159 (70.2)/18 (21.4), 1.967짹0.24 (66) P<0.001/P<0.001
281 (96.4)/3 (3.6), 2.216짹0.14 (81)81 (96.4)/3 (3.6), 2.255짹0.12 (81) P=1.0/P=0.0253 (63.1)/27 (32.1), 2.033짹0.24 (57) P<0.001/P<0.00149 (58.3)/27 (32.1), 1.964짹0.27 (57) P<0.001/P<0.001
1 vs. 2P=0.650/P=0.69P=0.650/P=0.692P=0.01/P=0.102P=0.225/P=0.559
Escherichia coli12112 (100)/0 (0), 2.287짹0.11 (12)12 (91.7)/1 (8.3), 2.268짹0.09 (11) P=1.0/P=0.2911 (91.7)/1 (8.3), 2.065짹0.19 (11) P=0.31/P=0.0029 (75.0)/2 (16.7), 1.923짹0.31 (10) P=0.06/P=0.003
212 (100)/0 (0), 2.294짹0.09 (12)11 (91.7)/1 (8.3), 2.199짹0.13 (11) P=0.31/P=0.048 (66.7)/4 (33.3), 2.023짹0.19 (8) P=0.03/P=0.0088 (66.7)/4 (33.3), 1.989짹0.18 (8) P=0.03/P=0.003
1 vs. 2P=1.0/P=0.829P=0.31/P=0.188P=0.132/P=0.025P=0.65/P=0.818
Enterobacter cloacae919 (100)/0 (0), 2.061짹0.09 (9)9 (100)/0 (0), 2.084짹0.12 (9) P=1.0/P=0.6058 (88.9)/1 (12.5), 1.945짹0.15 (8) P=0.30/P=0.0727 (77.8)/ (12.5), 1.885짹0.13 (8) P=0.13/P=0.001
29 (100)/0 (0), 2.106짹0.09 (9)8 (88.9)/0 (0), 2.032짹0.17 (9) P=0.30/P=0.1984 (44.4)/2 (25.0), 1.748짹0.34 (7) P=0.009/P=0.0325 (55.6)/2 (25.0), 1.807짹0.14 (7) P=0.023/P<0.001
1 vs. 2P=1.0/P=0.141P=0.30/P=0.449P=0.046/P=0.266P=0.0317/P=0.089
Klebsiella oxytoca515 (100)/0 (0), 2.268짹0.11 (5)5 (100)/0 (0), 2.271짹0.09 (5)3 (60)/1 (20.0), 2.048짹0.26 (4)3 (60)/2 (40.0), 2.187짹0.03 (3)
25 (100)/0 (0), 2.168짹0.03 (5)5 (100)/0 (0), 2.230짹0.10 (5)2 (40)/2 (40.0), 1.906짹0.40 (3)1 (20)/3 (60.0), 1.762짹0.25(2)
Klebsiella aerogenes414 (100)/0 (0), 2.180짹0.24 (4)4 (100)/0 (0), 2.281짹0.13 (4)3 (75)/1 (25.0), 2.277짹0.10 (3)4 (100)/0 (0), 2.231짹0.10 (4)
23 (75)/1 (25.0), 2.340짹0.07 (3)4 (100)/0 (0), 2.278짹0.17 (4)2 (50)/2 (50.0), 2.056짹0.22 (2)3 (75)/1 (25.0), 2.196짹0.05 (3)
Raoultella ornithinolyticaa)212 (100)/0 (0), 2.515짹0.01 (2)2 (100)/0 (0), 2.509짹0.04 (2)2 (100)/0 (0), 1.962짹0.08 (2)1 (50)/1 (25.0), 2.371짹0.00 (1)
22 (100)/0 (0), 2.482짹0.05 (2)2 (100)/0 (0), 2.470짹0.07 (2)2 (100)/0 (0), 2.461짹0.01 (2)2 (100)/0 (0), 2.155짹0.04 (2)
Citrobacter freundii111 (100)/0 (0), 2.200 (1)1 (100)/0 (0), 2.438 (1)1 (100)/0 (0), 2.073 (1)1 (100)/0 (0), 1.977 (1)
21 (100)/0 (0), 2.301 (1)1 (100)/0 (0), 2.311 (1)1 (100)/0 (0), 2.116 (1)1 (100)/0 (0), 2.261 (1)
Total1171115 (98.3)/2 (1.7), 2.213짹0.16 (115)114 (97.4)/3 (2.6), 2.300짹0.14 (114) P=0.9/P=0.2096(82.1)/16 (13.7), 2.017짹0.22 (101) P<0.001/P<0.00183 (70.9)/24 (20.5), 1.978짹0.24 (83) P<0.001/P<0.001
2113 (96.6)/4(3.4), 2.222짹0.14 (113)113 (96.6)/4 (3.4), 2.236짹0.14 (113) P=1.0/P=0.3172 (61.5)/37 (31.6), 2.015짹0.27 (80) P<0.001/P<0.00168(58.1)/37 (31.6), 1.965짹0.25 (80) P<0.001/P<0.001
1 vs. 2P=0.41/P=0.49P=0.70/P=0.22P<0.001/P=0.71P<0.001/P=0.38

Abbreviations: SD, standard deviation; IMP-MacConkey agar, MacConkey agar containing 1 關g/mL imipenem..

a)Raoutella ornitholytica was identified by MALDI-TOF, P-values of each results of 1 and 2 days were calculated by comparison of successful identification rates/score value of each media type to those of BAP on the same day of incubation, and P-values of 1 vs. 2 days results were calculated by comparison of successful identification rates/score values of 2 days results to those of 1 day on each media.


DISCUSSION

In this study, SIRs were significantly lower for colonies grown on Mac compared to those grown on BA and MH. The identification rate was further decreased when colonies on IMP-Mac were used. Because microbial identification by MALDI-TOF is based on unique protein signatures, certain media types and supplements may interfere with identification [6,8,9]. In a previous study, the type of media was found to affect the identification performance of MALDI-TOF MS for enteric gram-negative rods (EGNR), while growth on Mac showed similar performance as growth on BA, even when using the direct colony method [10]. Pseudomonas species are affected by growth on Mac more significantly when using the direct method described in the previous study. The reason for the difference in SIRs between media is unclear. The type of media is thought to affect the cell wall condition, making the colonies more mucoid in Pseudomonas species and affecting identification performance using the direct method [11]. This explanation may also apply to EGNR such as K. pneumoniae, which shows the greatest mucoid character and is the most prevalent CRE. Pigmented media such as Mac may affect protein spectral profiles, as has been observed for fungal pigments [12]. Additionally, bile salts in Mac may block ionization effects during examination [11]. In conclusion, species identification of EGNR by MALDI-TOF MS using the direct method was affected by the type of media used; growth on BA and MH is preferred to growth on Mac and IMP-Mac.

Colony aging also affected the success rate of identification when the colonies were grown on Mac. Colony aging from 5 to 48 hours did not impact identification performance [11]. Thus, very short-term incubation of 4 hours on solid media is sufficient to identify gram-negative rods, and further extending the incubation time does not improve SIR using MALDI-TOF [13]. Although the score value of correctly identified specimens did not change significantly with colony aging, the number of isolates showing no peak was significantly increased (Table 1). The effects of colony aging on BA and MH were not significant regardless of species, while results from Mac or IMP-Mac yielded lower confidence scores after 2 days of incubation compared to 1 day of incubation among E. cloacae and K. oxytoca. The bile salts present in Mac may interfered with the ionizing process because Hektoen Enteric agar or Salmonella-Shigella agar containing more bile salts showed lower confidence scores than MAC or xylose lysine deoxycholate agar [11]. The effect of aging on E. coli was lower on MAC than on K. pneumoniae when using the direct method [11]. This suggests that interference is species-dependent. Aging on Mac may cause more interference when using MALDI-TOF for identification than that when using the direct method. Considering that CRE surveillance culture is not considered as an urgent condition in most clinical laboratories, colony reading is often delayed during weekends or holidays. The aging effect on selective agar may be a significant limitation in the application of MALD-TOF to identify colonies on MAC or IMP-MAC.

A significant limitation of in this study was the small sample size, which did not include all common species of CRE. However, K. pneumoniae is the most prevalent species among CRE in Korea and showed a significant difference by media type and colony aging. The results demonstrated that growth on IMP-Mac certainly significantly reduces the ability of MALDI-TOF to identify CRE. In conclusion, for reliable species identification of CRE grown on IMP-Mac, a disk diffusion set on MH should be applied when using the direct method with MALDI-TOF. Further evaluation of larger sample sizes and a greater number of species is required to generalize these conclusions.

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