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Performance of MALDI Biotyper for Species Identification of Carbapenem-Resistant Enterobacteriaceae by Media Types and Incubation Time

Young Eun Cho, Byoung Hu Choi, Jeonghyun Chang, Heungsup Sung, and Mi-Na Kim Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea 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 μg/mL (IMP-Mac). CRElike 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 (2.017±0.22) and IMP-Mac (1.978±0.24) than for BA (2.213±0.16) (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. (J Lab Med Qual Assur 2018;40:155-160)


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 (Carbapenemaseproducing 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][6][7] score values of the identification results were analyzed for each media type and incubation time.

Statistical Analysis
The SIRs of each media type were compared by chisquare 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.

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 (  [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